Copper in biological systems. A report from the 6th Manziana Conference, September 23-27, 1990.

Copper in Biological Systems. A Report From the 6th Manziana Conference, September 23 -27, 1990 Helmut Beinert Department of Biochemistry and National Biomedical ESR Center, Medical CoUege of Wisconsin, Milwaukee, Wisconsin

ABBREVIATIONS

Enzymes and proteins: AO, amine oxidase; and as proposed in reference 3, BSAO, bovine serum AO; SSAO, swine serum AO; SKDAO, swine kidney AO; PSAO, pea seedling AO; APAO, arthrobacter PlAO; MADH, methylamine dehydrogenase; AAO, ascorbic acid oxidase; CY-AE, cr-amidating enzyme; AZ, azurin; COX, cytochrome c oxidase; CP, ceruloplasmin; DBH, dopamine P-hydroxylase; GO, galactose oxidase; Hc, hemocyanin; MT, metallotheonein; NIR, nitrite reductase; SOD, superoxide dismutase. Cofactors: Dopa, 3,4 dihydroxyphenylalanine; Topa, 3,4,6 trihydroxyphenyl-alanine; PLP, pyridoxal-phosphate; PQQ, pyrroloquinolinequinone. Reagents: DDC, diethyldithiocarbamate; DMG, diaminoguanidine; DMSA, dimercaptosuccinic acid; NTA, nitrilotriacetic acid. Technique-related: XANES, x-ray absorption near edge spectroscopy; EXAFS, extended x-ray absorption fine structure; ENDOR, electron-nuclear double resonance; ESEEM, electron spin echo envelope modulation; CD, circular dichroism; MCD, magnetic circular dichroism; NMRD, nuclear magnetic resonance dispersion; nqi, nuclear quadrupole interaction; DSC, differential scanning calorimetry.

The framework of this conference was very much the same as that of the fifth one [l] with one significant change: the sessions were held again in the now remodeled parco, or when the weather interfered, in the “Great Hall” of the Villa Giulia as in the old days. This brought back not only memories, but also some of the unique atmosphere of these meetings. However, the leisure and intimacy originally charac-

Address reprint requests to: Professor

Bruno Mondovi, Dipartimento di Science Biochimiche, Universid di Roma “La Sapienza,” Piazzale Aldo Moto, 5, 00185 Roma, Italy, and in the USA to Professor Helmut Beinert, Department of Biochemistry and National ESR Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, US. Journal of Inorganic Biochemistry, 44, 173-218 (1991) 0 1991 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, NY, NY 10010

173 0162.0134/91/$3.50

174

H. Beinert

terizing these conferences could not be recovered with some 80 participants now milling about, but the ground covered and the depth of the discussions clearly gained with so many minds involved. As five years had elapsed since the previous conference, there was. of course, a considerable shift in population, with some new and mostly younger faces appearing and some of the old-timers missing. These meetings are a fairly reliable mirror of the trends in the biochemical and related sciences. Thus, for instance. while at previous meetings all those present listened with awe to the only crystallographer present who could show a complete structure of a copper protein, at the 1990 meeting there were four crystallographers, three of them discussing new structures of copper proteins. It was also the first time that molecular biology and genetics made conspicuous and greatly appreciated inroads into meetings that were traditionally almost dominated by spectroscopists, inorganic chemists, and the more physicochemically oriented [cf. 21. The production of specific mutants of proteins and the ready availability of amino acid sequences deduced from DNA sequences clearly showed their impact on the field of copper proteins. In order to keep this report at a manageable size I will use the strategy of presenting in more detail those contributions of which there ia yet no reasonably accessible, published record available, while I will mention only the topics covered and the principal conclusions reached, together with the most recent references. foi those where publications exist. The distribution of handouts by the speakers was a great help; and with the idea that I could not say things better than the authors themselves, I took the liberty of lifting generously from these handout!,

AMINE

OXIDASES The first session, dealing with amine oxidases, was awaited by many with some suspense because of recent developments in this field [4]. B. Mondovi gave a brief survey of what can now be considered as reasonably established concerning these enzymes, and what is still in ddubt [S]. It is not yet clear whether the copper in these enzymes undergoes oxidation-reduction during catalytic turnover (see however. Dooley, below). There is increasing evidence that the two copper ions per subunit are not equivalent, although this is not obvious from EPR (except for swine serum amine oxidase). The cardinal question, however, at this time is the nature of the undoubtedly present organic cofactor. While for many years pyridoxalphosphate had been thought (never unambiguously proven) to be the cofactor, at the last conference in 1985 PQQ was the favorite candidate and most participants of that meeting had expected that it would be only a matter of time until final corroboration of this was forthcoming. While in the meantime much circumstantial evidence for the presence of PQQ in amine oxidases appeared in the literature, no definitive identification was reported. However, by mid-1990, evidence for 6-hydroxy-Dopa being the organic cofactor was published 141, evidence which one can reasonably consider definitive. Mondovi felt that there still remained the question as to the stoichiometry of Cu vs cofactor, whether 1: 1 or 2: I. In BSAO, for instance. only one carbonyl group is titratable with phenylhydrazine-the usual test for the carbonyl-containing cofactor --while the presence of a second, less reactive carbonyl becomes apparent with the use of some hydrazide derivatives [6]. This raises the question whether an additional organic prosthetic group is present or whether hydroxy-Dopa is able to react with two equivalents of carbonyl reagent. Progress toward understanding the reasons for

COPPER IN BIOLOGICAL

SYSTEMS:

MANZIANA

REPORT

175

this apparent discrepancy should be possible by studying the ratio of copper vs active site prosthetic group in purified preparations from different laboratories [7]. J. A. Duine then went over the history of the development of methods for the detection of PQQ, some of them based on the reactivity of carbonyl groups with phenylhydrazine, a reaction that had been used for the detection of the organic cofactor in amine oxidases long before PQQ was known. Duine’s contributions to this subject can be found in references 8-11. Duine’s conclusion from the application of these methods to a number of enzymes had been that the cofactor in these proteins was covalently bound PQQ and this had received support from a number of laboratories using his or other chemical and also spectroscopic methods. Duine concludes now from a consideration of the most recent results in various laboratories and in his own that 1) strong evidence exists for the presence in amine oxidases of a covalently-bound organic cofactor of quinone character and 2) the hydrazine method is not selective since it is now known, for a number of enzymes which had been thought to contain covalently-bound PQQ, that they have unique amino acid residues that react with hydrazine, as, e.g., methylamine dehydrogenase, galactose oxidase, and dopamine-/3-monooxygenase [ 12- 141. Apparently, conditions during or after derivatization determine the final product. Duine emphasized that it was necessary in every case to isolate the product. But even if PQQ can be extracted, it may not be the compound originally bound. It will have to be determined what the reactions are that convert the originally present groups to PQQ. To Duine, 6-hydroxydopaquinone now seems a good candidate for being the cofactor of the amine oxidases of quinoprotein nature. Duine said, however, that he finds only one cofactor molecule per dimer, whereas Klinman finds two and he thus considers the stoichiometry not yet settled. Mondovi seconded this statement. Duine also commented that Topa dinitrophenylhydrazone can be distinguished from PQQ phenylhydrazone by chromatography and that the spectra of quinoproteins are distinctly different from those of PQQ. In a comment on his immunological approach to identify the cofactor in amine oxidase, Finazzi-Agro pointed out that the epitope of PQQ is not known and that even monoclonal antibodies react with several different proteins. The conclusions by Duine were an important contribution, as they open the way for a reconciliation of a number of conflicting results and statements in even the most recent literature on the subject. A brief publication emanating from this very meeting will state clearly the consensus reached by those present and active in this field [7]. Unfortunately, it appears at this time that the results reported by Franca Buffoni in the next lecture cannot be reconciled with the picture that has emerged from the discussions. Publications germane to the subject, which have appeared after the meeting can be found in references 15-19. Next, Franca Buffoni reported on her studies on a novel inhibitor with selectivity for tissue-bound semicarbazide-sensitive amine oxidases, as reported in reference 20. Following this, she drew attention to her continued work on the identification of pyridoxalphosphate (PLP) as a component of pig benzylamine oxidase. A new procedure for the isolation and identification of PLP in proteins has been published involving gas chromatography-mass spectroscopy [21]. By this method PLP was identified as a component of pig benzylamine oxidase in a stoichiometry of 0.75-l mole of PLP per mole of enzyme [22]. PQQ was not found and what appears as PQQ on hydrolysis had not been PQQ originally. Buffoni stated, however, that PQQ may still be there, but cannot be identified by the approaches used; also, other cofactors are not excluded. No work has been done yet in Buffoni’s laboratory on

176

H. Beinert

identifying Topa; however. she has analyzed a preparation of BSAO supplied by Klinman, with results analogous to those reported on benzylamine oxidase prepared in her own laboratory. Taking these results at face value one could ask whether it is possible that PLP might be present in addition to the other cofactor (see above). However, the stoichiometry alone. of groups reacting with carbonyl reagents, speaks against this, and, particularly, as J. P. Klinman pointed out in the discussion, her experiments on “reductive trapping’* [23. 241 with “C labeled substrate and ‘H labeled NaCNBH i, rule out a single carbonyl as the reactive species. but are compatible with a dicarbonyl compound as the target. In addition, Knowles commented that he was unable to detect PLP in SSAO bq “P NMR. In view of the numerous conflicting results in this area, it would seem desirable that the occurrence of PLP in amine oxidases be confirmed in independent work. Following Franca Buffoni, Judith Klinman presented her pioneering work. as recently published. on rhe identification of h-hydroxy Dopa (or Topa) as the cofactor of SSAO 141. More recent work on peptides isolated after thermolysin digestion of the related amine oxidases from yeast, pig serum, kidney. and pea seedlings yielded analogous hexapeptides (xx-cofactor-xxx) containing the cofactor. Kiinman also presented evidence that there is no half-of-the-sites activity m her preparations. She routinely finds two phenylhydrazines hound per enzyme dimer using cn?ymes of high activity. D. M. Dooley opened his talk with the challenging statement (see Mondovi. above) that the copper in amine oxidases is reduced by substrate after all [25t. It had been known that copper can be reduced by substrate in the presencr of cyanide. and in the plant enzymes it can be reduced even in the absence of qanide. Dooley has identified distinct features in the light absorption spectrum around 350 nm that coincide with reduction and are intensified on cyanide addition. He has now observed that these features are very temperature dependent. disappearing r)n cooling, which suggested that reduction of Cu is not observable by EPR on frozen samples for this very reason. On examination of various amine oxidases by room temperature EPR, minor decreases in the broad Cu signal were seen concomitant with the appearance of a sharp free radical type of signal at g = 2. when substrate wa:, added anaerohitally. Fortunately. two c,f the enzymes, those from Grthrobacter PI and pea seedlings. showed as much as a 30-40% reduction of the Cu signai when at room temperature, while the radical signal that appeared could account for about 20% of the oxidizing equivalents so generated. These results were confirmed by CD spectroscopy which can also be carried out at room temperature. On admission of oxygen the system became instantly reoxidized. Detailed kinetic studies have not yet been done. On the basis of these observations Dooley proposes a catalytic cycle as shown in Scheme I. He further presented Resonance Raman spectra of the phenylhydrazones of BSAO. SKDAO, APAO, and PSAO which left little doubt that the cofactor in at least the first three is identical. He then showed comparisons of the Resonance Raman spectra of the phenyl- and nitrophenylhydrazones from WSAO versus those of the BSAO-derived peptides containing the cofactor, as wll as :I comparison of spectra of these derivatized peptides with those of Topa phenyl- and nitrophenylhydrazone and with PQQ dinitrophenylhydrazone. These spectra were cc)nvincing that Topa and not PQQ is the cofactor in BSAO and the closely related emymes. In the ensuing discussion it was pointed out that the scheme shown. while very reasonable for the equilibrium situation studied, may not describe the events occurring during turnover. Similar situations have been encountered in father enzymes, where metals


pox E\

RCH2NH2

\

Cu(ll)

Ii202 +NH, 2H- I 7 E\ [

cu(ll)-op-

I

I/

RCHO

_

SYSTEMS:

MANZIANA

REPORT

177

SCHEME 1. Possible catalytic cycle of copperEPred containing amine oxidases. The species in brack\ CU(ll) ets is a hypothetical intermediate, shown here to emphasize the possibility of sequential one-electron steps in the reduction of 0,. Q,, = oxidized quinone, Q’ = semiquinone, and Qred = twoelectron reduced quinone. (From reference 25, reprinted by permission from Nature 349, pp. 260-264; copyright 1990, Macmillan Magazines Limited.)

and a quinone-semiquinone-hydroquinone system are potentially involved, as, e.g., in some flavoproteins. P. F. Knowles drew attention to the fact that SSAO differs from other amine oxidases in some of its properties. Thus SSAO is the only amine oxidase thus far for which a small but significant difference between the two cupric ions can be detected by 35 GHz EPR [26]. Furthermore, the use of a new assay procedure shows that there are two molecules of quinone cofactor bound per molecule of protein in SSAO; only the more reactive of these quinones is required for activity [26]. By contrast, in BSAO, both quinone cofactors seem to be essential for activity (see above). Other differences between the amine oxidases are indicated by studies of the magnetic field dependence of the relaxation rate of solvent protons, i.e., Nuclear Magnetic Resonance Dispersion (NMRD) studies. The results suggest that one. ligated water molecule per copper in rapid exchange with solvent is present in all amine oxidases studied (BSAO, SSAO, SKDAO, and APAO) but that an additional water, probably equatorially coordinated, is present in SSAO and APAO [27]. The incorporation of copper into copper-depleted (“apo”) BSAO and SSAO was also reported [27]. For both enzymes, the relaxivity (i.e., the total relaxation rate per mM of protein), the specific activity, and the intensity of the principle visible absorption band are all approximate linear functions of the copper content. This conclusion differs from an earlier report [28] that the single-copper form of BSAO is inactive and suggests that the procedure for reconstitution with copper is critical. Morpurgo et al. [29] had ascribed this difference to the regime of copper readdition. When the cofactor of apo BSAO was reoxidized before adding copper, an inactive single Cu containing enzyme was produced [28]. Finally, Knowles reported that substrate amine has no effect on the NMRD profile of BSAO, suggesting that the substrate does not bind to copper. By contrast, NMRD studies show that addition of alcohol substrate to inactivated galactose oxidase [30] displaces the single outer sphere water [18] which is consistent with substrate binding to copper. Laura Morpurgo reported on interactions between copper and the carbonylcofactor. These were studied via the effect which modification of the copper sites exerted on the reactions of the carbonyl with hydrazines and hydrazides. It had been shown in previous work that copper and carbonyl are not very close to each other, as copper was able to bind dithiocarbamate (DDC) at the same time as phenylhydrazine was attached to the cofactor; however, the properties of each adduct were modified when the other inhibitor was also bound [29]. It had also been concluded that Cu is not directly involved in the catalytic oxidation of the pseudo-substrate benzylhydrazine, since Cu(I) species were not detected and identical reaction products were obtained slowly when DDC was present, which irreversibly binds to the Cu ions. It

178 H. Beinert

was therefore suggested that oxygen does not react at the copper site. but at the carbonyl site, forming a hydroperoxo adduct [31]. This idea received support from the observation that the reaction was even less affected when Co-substituted BSAO was used. This derivative showed about one-half the usual benzylamine oxidase activity [32]. In further work the reaction of BSAO with a series of hydrazydes of the general formula R#J(CH~),,CONHNH, was studied. in which R is a phenyl substituent para to the hydrazide residue [33]. When n -= t and R uas a second aromatic ring, two mol of the compound were bound per rnol of dimer. the first one rapidly, the second one very slowly. When n ==0, only one mol reacted per mol of dimer. Phenyihydrazine was able to find a second carbonyl in both adducts, displacing one n = 1 hydrazide when two were bound. This indicates that the slowI> reacting molecules of the n = i compounds had reacted tvith Y sec,ond cofactor carbonyl. Phenylhydrazine. therefore. seems to react with only one--half of thr available sites on the enzyme reducing the cofactor at thi\ site to a hydroqumone form. However, binding can occur at both sites when a hydrophobiti interaction between the hydrazide R group and the protein prevents clc,nformational changes following the first binding. the C’u-cofactor interaction. With the same goal. namely that of investigating S. Suzuki studied electronic and CD spectra of BSAO after reaction i)f’ the enzyme with phenylhvdrazine and aminoguanidine (.4G), which oni) modify the cofactor. The 475 nm-absorption band due to the organic cotactor is decreased when the substrate benzylamine is added anaerobically, indicating the formation of the reduced cofactor. The corresponding CD bands near 370 and 450 nm also disappear. whereas the 650 nm CD band in the region of the d-d transitions of the Cut II) center appears to shift to about 7 10 nm. After treatment with phenylhydrazinc BSAO sht ws intense absorption and CD bands around 4.50 nm. In the region hetwecn 500 md 800 nm. where Cu(II) d-d transitions are expected. the absorptil)n and (‘II> hanjs shift tc,ward the red. Particularly the CD band near 7 10 nrr, is rather simiiar io that tli‘ USA0 after reaction with benzylamine. AC&treated BSAO shows intcnsc ah t!~ binding ot substrate or the inhibitor. Suzuki suggests that the Cu(II) center ~txhibir~ng the 720 nm CD band is in a .*rc;tdy“ state where the coppe! ian ~juickiv :I-ansfer two electrons from the reduced cofactor to dioxygcn. Suzuki’s presentation was followed by a brief report h), J. c‘. P&l-son on the observation of radicals from PQQ and 6-hydroxydopa by EPR and their comparison with radical signals seen with A0 from lentils. These signal> can be produced in the absence of cyanide (see above). Pederson finds a great diffcrencc hctwecn the enzyme- and PQQ-radical. as one would now expect after the foregoing. The linewidth of the Topa radical itself was much too narrow to account for the enzyme spectrum: however. Topa reacted spontaneously with putrescine and other enryme substrates to give an amino-Topa radical. The !,imulation of this radical under immobilized conditions resulted in a spectrum ver\’ similar tin that ot the cnzymc radical.


SYSTEMS:

MANZIANA

REPORT

179

Anna Giartosio then presented results from her studies on several Cu-proteins by differential scanning calorimetry. With the blue oxidases investigated, namely laccase, ascorbate oxidase (AAO), and ceruloplasmin (CP), a single peak was observed in the thermograms, which could be resolved into 2-4 two-state transitions in the holoenzymes and into two transitions in the Cu-depleted enzymes. However, type-2 depleted lactase and ascorbate oxidase showed thermograms indistinguishable from those of the holoenzymes, indicating that type-2 Cu has no structural function in these proteins. Type-l and type-3 Cu ions, however, have a noticeable influence on the stability of these proteins [34]. The thermogram of amine oxidase (BSAO) shows a quite different pattern, characterized by three well separated peaks, which correspond to four nearly independent two-state transitions. Correlation of enzymatic activity and Cu content with the integrity of the calorimetric transitions allows one to attribute these transitions to definite protein domains [35, 361. With this report the chapter on amine oxidases was closed, but not that on the role (or absence) of PQQ in several other proteins.

GALACTOSE

OXIDASE

In the paper following this report, J. W. Whittaker presented the fascinating story of yet another enzyme that had been a candidate for PQQ involvement. This Cuenzyme, namely galactose oxidase (GO) from the fungus Dactylium dendroides, has a long and indistinct history at the Manziana meetings, as there was little progress apparent and to most the problem seemed rather intractable and consequently unattractive. Not so to the Whittaker family, and what was presented at this meeting leaves little, if anything, left to clean up. Fortunately, the crystal structure was also solved to high resolution at the same time (see next report), so that the story of this enzyme has become a textbook example as to the conspiracy of nature against unsuspecting researchers on the one hand, and the power of logic, persistence, and the application of the proper technique at the right time, on the other. The crux was that the enzyme, as obtained on isolation, could be shown to be a mixture of oxidized active and reduced inactive forms with the somewhat paradoxical twist that the form that showed the long known type-2 EPR signal of Cu(I1) originated from the latter, namely the reduced form. However, what was reduced in this inactive form was not the copper but an organic cofactor, the same that had been suspected to be PQQ. The interested reader is referred to the original work on the subject for the details of this very interesting and instructive research adventure, which is available in the literature of 1988-90 [30, 37, 381. The active enzyme, which contains the “cofactor” in the free radical state shows neither the typical EPR properties of Cu(I1) nor those of a free radical, since there is a strong antiferromagnetic exchange interaction (2 200 cm -I) between both of these components, which requires direct coordination of the radical to the copper. Present evidence, supported by the crystal structure, strongly points to a modified tyrosine residue as the site of the radical. Dopa was not detected in the protein. The closely related subsequent paper by N. Ito on the 1.7 A crystal structure of galactose oxidase [ 181, no less exciting than the preceding one, presented an unusual structure of pseudo 7-fold symmetry, evidence of a cysteine-tyrosine bridge, and a stacking interaction of these residues with a tryptophan. This constellation of amino acids may well represent the “radical” generation portion of the “cofactor” site. Ito described GO as consisting of three predominantly &structure domains with only a

180

H. Beinert

FIGURE 1. Computer generated ribbon diagram of the second domain of GO, looking along the pseudo 7-fold axis. There is a large hole along the axis, which is filled by two @ strands from the third domain and a number of water molecules.

single short a-helix. He sees in this preponderance of @-structure one of the reasons for the great stability of the enzyme. The first domain, including residues l--155, represents a P-sandwich structure and is linked to the second domain, which includes residues 156-532. by a well ordered stretch of polypeptide. The second domain is the largest domain and has pseudo 7-fold symmetry, described by Ito by the model of a 7-petaled flower. where each petal consists of a 4-stranded anti-parallel P-sheet. The cupric ion is found on the solvent-accessible surface of this domain in the vicinity of the ‘I-fold axis. The third domain comprises residues 533 -639 and is located on the opposite side (with respect to the second domain! of the copper. Two of the seven P-strands in the third domain quasi pierce the middle of the second domain along the pseudo ‘i-fold axis and furnish a ligand to the copper ion (Fig. 11. The void between this finger-like structure and the second domain is occupied with well-defined water molecules. No density was seen indicating either the presence of carbohydrate or of PQQ. The cupric ion is surrounded with a number of aromatic side chains. The copper site has five ligands in square pyramidal coordination (Fig. 2). Tyr 272, his 4967. his 581. and an acetate ion form an almost perfect square with the fifth axial ligand. tyr 495. in the absence of acetate this ligand ion is replaced by a small molecule, presumably water, at the unusual distance of 0.28 nm. Now focusing on the possible workings of the enzyme. we note that one of the copper ligandb, tyr 272. is covalently bound at C, to the sulfur atom of cys 228, in a bond that seems to have partial double-bond character, while C,, of cys 228 lie?, in the same plane as does the ring of tyr 272. The indole ring of trp 290 is stacked on what Ito calls this “pseudo-side chain. ” On the basis of this model Ito suggests that a free radical associated with this tyrosine is involved as the secondary copactor rather than PQQ. Ito sees an essential role in the catalytic mechanism for the unusual bond between cys 228 and tyr 272. The sultir is thought to assist in delocalization of the free radical. while the stacking trp provides stabilization.

His 496

His 58, N

N-

,’

‘,,?_/

AA \ o ~~ .__~ .~ ~~ Acetat*> ion

o ,Tyr

,’ 27?

FIGURE 2. Schematic drawing of the coordination of Cu in galactose oxidase according to ref. 18. (Reprinted by permission from Nature 350. pp. 87 -90. 1991. Macmillan Magazines Limited.)


SYSTEMS:

MANZIANA

REPORT

181

SCHEME 2. Reaction mechanism proposed for C. violaceurn PAH by Benkovic .

PHENYLALANINE

HYDROXYLASE

The first day’s sessions came to an end with a presentation by S. J. Benkovic on phenylalanine hydroxylase (PAH) from Chromobacterium violaceum, one of the tetrahydropterin dependent aromatic amino acid hydroxylases that include tryptophan and tyrosine hydroxylases. The tetrameric mammalian enzyme contains one Fe per monomer, whereas the monomeric bacterial enzyme from C. violaceum contains a single Cu(I1) ion of type-2. Reduction of PAH, i.e., that of its Cu(I1) ion, is a prerequisite for catalysis. 6-Methyl-tetrahydropterin in the presence of the phenylalanine is the natural activation system [39, 401. The reduction of the metal appears to be the rate limiting step with a conversion of axial Cu(I1) to linear or tetrahedral Cu(1) as the rate limiting ligand rearrangement. The question as to the proximity of the Cu ion to the pterin and possible binding sites was approached with EPR techniques including ESEEM and site specific isotopic labeling with 15N [41]. It is concluded from this work that Cu is bound to pterin through the 5-N and to two imidazoles from histidines. Steady-state kinetic analysis indicates a partially ordered mechanism of the kind shown (Scheme 2). E-O, has been trapped in rapid quench-chase experiments. Approximately 60% of the expected 0, was found and magnetic susceptibility measurements indicated that the magnetic moment of 0, is diminished, which is consistent with formation of Cu(II)-0, [42]. Pterins do not react rapidly with 0, so as to allow binding of 0, to the enzyme. As in other instances at this meeting, sequence comparisons made interesting contributions [43]. Thus Benkovic could compare the sequences of the Cu enzyme he was reporting on with sequences available for rat and human PAH, which, as stated above, are both Fe proteins. The overall sequence homology between the Cu enzyme and the two mammalian ones is 24 % . However, a highly conserved region (amino acids 77-152) is analogous to the 35 kD catalytic domain of mammalian PAH with 36% homology (Fig. 3). It is thus noteworthy that nature utilizes the same overall protein structure, but chooses alternative metals. It is an

.

4&L

A

A

AA

,

1A

1

A

FIGURE 3. Alignment of amino acid sequences for C. violaceum, rat and human PAH. The numbering is that of the C. violaceum enzyme (from reference 43). Residues in boxes are identical, A indicates those residues that are also identical in trp and tyr hydroxylases.

182

H. Beinert

interesting question what may determine such a choice. Benkovic raised hopes that a crystal structure may be available before too long.

DOPAMINE

&MONOOXYGENASE

N. J. Blackburn started the second day of the meeting with a report on the enzyme dopamine &hydroxylase (dopamine /3-monooxygense, DBH). a topic raising several controversial points. While there was no more doubt among those present that DBH has two copper ions per monomer within a tetramer containing eight Cu ions (cf. 198.5 meeting). the question of the ligand environment of the Cu ions is not unambiguously answered and differences between the ions arc difficult to discern because selective removal is not readily accomplished with two ions of closely similar dissociation constants. Much of the discussion centered around the results of EXAFS studies done on the same enzyme (made by the same purification procedure) albeit by different people and from different animals although the same ,pecies. with the XANES and EXAFS carried out in different countries and by different collaboraring colleagues. Blackburn’s group came to the conciusion that the most likely copper environment for the oxidized form consists of maximally 3 histidincs and one or two water molecules in addition in a site of close to tetragonal symmetry. in the reduced form two histidines move in closer and thcrc appears a heavier-,atom icatter in the Iigand shell 14.41: such as an S or Cl iigand. Blackburn prefers the rdea of 311 S atom furnished by a neighboring methionine. since there is no spectroscopic- or chemical evidence for thiol or thiolate in the vicinity of the Cu-sites. He was ahlc to USCto advantage the observation that CO binds to the reduced enzyme. and only to one 01 the two atoms present per monomer. Blackburn prefers the picture of two his. one met sulfur, and CO hound to the Cu(I) ibrm {Fig. 1-j. The behavior ot the two-copper site of DBH, writh a single CO bound: reminds us of the_:Cu-site in hemocyanin. However, the hemocyanin Cu-site with CO bound shows tluorescencc. whereas the DBH Cu-site does not which has been exploited experimentally, fluoresce. Blackburn concludes from this observation that. contrary to the situation with hemocyanin, where CO binds to both Cu ions present. DBH has a mononuclear CO-binding site. This is in line with the failure to observe a Ct.&u scattering by EXAFS. However, since in deoxyhemocyanin only one of the two fu-ions of the active site binds CO, a finding which is thus far not weti understood. the observed stoichiometry of CO-binding to reduced DBH cannot unambiguously rule out the existence of a binuciear Cu(1) site in DBH. As one would expect, CO inhibits DBH. A publication which describes the interesting use of CO as a probe for the Cu-site in DBH appeared just at the rime of the meeting [4Sl and a more rxtensivc paper IS in press [46]. After Blackburn’s lecture, J. J. Villafranca presented his views on DBH, which in a few, but nevertheless significant ways differed from those just hzard. Villafranca’s group has focused in the iast several years on the characterization of tht Cu sites by x-ray absorption and spin echo EPR spectroscopies. The spin echo results showed that the Cu environment is best described as having three to four ligands per Cu. with two magnetically distinct populations, i.e.. different N-Cu couplings 1471. From experiments in which the Cu depleted enzyme was reconstituted in steps with Cu it was concluded that the environments were similar whether the tetramer had 3. 4. or 8 Cu bound. X-ray absorption spectroscopy demonstrated that the Cu(I1) state as well as the Cu(1) state both had 4 N or 0 ligands. and that the Cuil) state had no S lipand


H20,

Met-$--l

I

,His

MANZIANA

REPORT

183

.x

Cd

His’

His

SYSTEMS:

‘His

)

\&I, His’

‘Hz0

I

Ascorbate

,His ,CU’

HIS co I

I cu,H’s1

HIS,

/

HIS

FIGURE 4. Reaction chemistry of DBH as suggested Blackburn from the experiments discussed.

by

[48] as reported by Blackbum and his collaborators [44]. Villafranca suggested that the observed heavy atom ligand, assumed to be S, may in fact be Cl-, since 1 mM Cl- was present in the preparation used by Blackbum, whereas Villafranca purposely avoided such potential heavy atom ligands. He then discussed his group’s work on the organic cofactor, which had been thought to be PQQ [14]. Villafranca pointed out that the enzyme used in his work had the highest specific activity ever reported for DBH and that according to spectroscopic and peptide analysis no PQQ is present in the freshly isolated enzyme. The peptide consensus sequence established by Klinman’s group for the Topa bearing peptide was not found in DBH. However, experiments with the two mechanism-based inhibitors phenylhydrazine and 6hydroxybenzofuran implicated his 249, tyr 477, and arg 503 as active site residues [49]. Arginine is in fact destroyed in the process of the inactivation of DBH. In the course of these investigations the location of the -S-S- bonds in the native enzyme was established and it was shown that there are two -S-S- bonds joining an enzyme dimer. The connectivity of these inter-dimer bonds is not yet resolved. It was also found that there are distinct glycosylation sites present and their location was determined [SO]. Finally Villafranca reported on his attempts to produce a half-met form of the Cu site. The enzyme is readily reduced by ascorbate and no EPR signal is seen in this reduced form. However, on exposure of reduced DBH to NaNO, and 0, the signal of Cu(I1) reappears-approximately one Cu(I1) per two Cu bound. This process is slowly reversible. The Cu(I1) signal does not appear if there is no 0, present. As was to be expected, this talk was followed by a lively discussion. One of the main items of contention was the number of his ligands in reduced DBH, of which Blackbum finds less than three. He emphasized that the CO chemistry which he observed (see preceding lecture) speaks for low histidine coordination. Curiously,

184

H. Beinert

most interpretations of either group are based on EXAFS data. There is indeed a difference in the EXAFS spectra of the reduced enzymes. whereas the spectra of the oxidized forms appear identical. Hasnain pointed out that the difference in the results deduced from EXAFS data could very well also be due to the method of data evaluation, with one group (Daresbury) using multiparameter fits and the other (Stanford) fits to model compounds. Peisach suggested that the twc! groups exchange preparations. which, according to the method used, were expected to be identical. Concerning the organic cofactor in DBH, Duinc remarked that by his methods elaborated for the determination of PQQ. he finds what he considered to be PQC.! in stoichiometric amounts in DBH. It always is a particular bonus to the enzymologists present, when during such a meeting a new enzyme surfaces, and particularly one that catalyzes an unusual reaction. This was amply the case in the sessions that followed. 13. Merkler discussed his studies on the peptidylglycine tu-amidating i-nzymc ior monooxygenasc) from rat medullary thyroid carcinoma ( CYAE). crAE catalyze> the conversion of C-terminal glycine-extended peptides to the corresponding C-terminal cu-amidatod peptides and glyoxylate. The reaction requires two reducing equivalenr>. most like11 furnished by ascorbate in vivo. molecular oxygen. and enzyme-bound Cu. Rat. 75 kDa OZAE has been cloned and expressed in mouse C113” cells 151. 521. From preliminary experiments on the purified recombinant wAE. d atoichiometr> ot‘ I --ii atoms of Cu(I1) per molecule of enzyme can be deduced. The purified (uAE will amidate any peptide-x-gly substrate [.53] and can accept electrons from a variety of with different reducing agents. The enzyme also shows a “reaction inactivation” ascorbate as the reductant. Protection against this inactivation is provided orIll- with reagents that react with H,02 and not with reagents such as superoxide dismutase or radical scavengers. Peptidyl-cY-hydroxyglycine has been suggested as a possibie intermediate in the amidation reaction. The notion has also been maintained that the conversion of this intermediate to peptidyl-NH2 ma) be catalyzed b> a separate enzyme. By the use of their recombinant enzyme. howcvcr. hlerkler and hih colleagues have been able to eliminate this possibility. Their parent;tl Cl27 cells showed only trace levels of amidating and lyasr activities. while the cicmes hhowned both activities, establishing that fadrenal medulla,


SYSTEMS:

MANZIANA

REPORT

185

pituitary, pancreas, and cardiac atria [57]. Nalbandyan finds one type-2 Cu per molecule in these enzymes. He also could show the close similarity, if not identity, of the physicochemical as well as antigenic characteristics of the crAEs from these various sources. They were encountered in granules in soluble and in membranebound form. Nalbandyan also proposed a possible mechanism for the regulation of these enzymes [58]. There is found in brain and in different kinds of secretory granules a very acidic protein which can occur as copper protein a well as apoprotein. This protein is able to remove Cu from either (r AE or from DBH and could thus in vivo modulate the activity of these enzymes [59].

NITRITE

AND NITROUS

OXIDE

REDUCTASES

The meeting now moved on to enzymes involved in the metabolism of simple nitrogenous compounds. Elinor Adman presented the 2.3 8, x-ray structure of nitrite reductase (NIR) from Achromobacter cycloclastes, a copper protein. This enzyme had been reported to be a dimer of 69 kD (per dimer) containing three Cu atoms per dimer. A small type-l blue copper protein, cupredoxin, is the electron donor to this enzyme. One may visualize the combination of cupredoxin and NIR as being analogous to lactase with four Cu per molecule. NIR crystals have one monomer per asymmetric unit [60]. According to the crystal structure [61] the molecule is actually a trimer, associated around a crystallographic threefold axis. There are two metal atom sites per monomer, one characteristic of a type-l blue Cu with 2 his, 1 cys, and 1 met as ligands, and the other metal site characteristic of a type-2 Cu with three his and a small ligand, probably OH. The Cu sites are 12 A apart and are ligated by adjacent residues in the sequence, his to the type-2 Cu, followed by cys to the type-l Cu. The polypeptide-fold of domain I and domain II is a Greek-key /3 barrel, as it is for other type-l blue proteins. Two of the ligands to type-2 Cu are supplied by domain I, the third comes from domain II of a neighboring molecule in the trimer. Superposition of domain I on the type-l domain of ascorbate oxidase (AA0 domain III) puts the type-2 Cu in the same place as the trinuclear cluster of AA0 (Fig. 5). The amino acid sequences of domains I and II of NIR are homologous and are related to those of cupredoxins. Considering homologies among Cu proteins, we may recall that Messerschmidt and Huber have shown that AA0 has homologies with lactase and ceruloplasmin [62] and Adman pointed out that the similarity of the NIR and AA0 structures suggests that the trimeric arrangement of NIR can serve as a plausible model for the spatial arrangement of the six ceruloplasmin domains [63].

FIGURE 5. Copper binding sites of ascorbate oxidase (top) and nitrite reductase (bottom). In each case the type-l site is in the upper left. In ascorbate oxidase the trinuclear Cu site is formed between domains on the same monomer, and in nitrite reductase between domains of d#krent monomers.

186 H. Beinert

One could consider the Cu site of NIR as an evolutionary precursor of that of the blue Cu oxidases; in other words, nature may have built up this site by adding two more Cu binding sites. Referring to most recent work, Adman commented that on soaking the NIR crystals with nitrite, new electron density was seen at the type-2 site. With repeated glimpses emerging into structure relatedness and evolution of molecules, these last sessions were among the high points of the meeting for many. P. M. H. Kroneck next presented studies on N,O reductase of PseudomonclJ stutzeri. He introduced this enzyme to the Manziana audience at the 1985 meeting While progress has been made on a broad front. as documented in recent publications 164. 651, the question as to the nature of the eight Cu atoms in the enzyme still is not satisfactorily answered. The enzyme has been obtained in a number of forms, which helped to deduce some properties of the coppers present. Among these forms four copper atoms can be is an apoprotein, from which a protein containing reconstituted; the EPR properties of this reconstituted form are \erq similar to those of a protein obtained from a mutant deticient in chromophore synthesis. The protein from the mutant has two coppers. In both of these forms, which are catalyticalil inactive, 50% of the Cu is EPR detectable and the hypertine pattern indicates Cu-Cu interaction, which is yet more clearly indicated in the EPR spectrum of the active X-Cu protein. On anaerobic reduction a catalytically incompetent. blue form of the enzyme is obtained, which shows a broad featureless EPR signal. It seems, therefore, that the Cu atoms required for catalysis may be as few as two and that much of the remaining Cu is present in the form of an interacting system of unknown structure and function. as it becomes manifest in the blue form. The EPR hyprrfine pattern and the quantity of Cu represented in the Cu EPR slgnal typical of the active forms suggests the presence of a mixed valence binuclear Cu pair. Along lines that became more and more apparent in previous papers of this session. one of the most interesting aspects of this work is the undeniable relationship that N:OR shows to cytochrome c oxidase ICOX). Such a relationship becomes more plausible ii’ we consider COX not as cytochrome oxidase but as an 0, reductase. as opposed to N,O reductase. Not only are there clear homologies in typical c’u binding sites (cys, his. and met ligands) in COXs from various sources and in N,O rcductase. but the EPR hyperfine pattern of the Cu signal, as far as it is not obscured hy the low spin heme signals in COX, is amazingly similar in both types of enzymes (661. May it hc recalled here that in COX only one of the two Cu atoms that habc traditionally been considered to be components of COX is EPR detectable in the resting (as isolated) enzyme, named Cu,. There is very strong evidence that the other Cu atom. named Cu,, is in close proximity ( e-3 ii) to one of the hemes (that oi cytochrome a3). with which it forms an antiferromagnetically coupled pair. Thus the question arises: if there is a mixed valence Cu pair in COX, where is the second copper’? However. there have been developments in the past few years that might furnish a tentative answer to this question. Careful analytical work in several laboratorirh on a number of types of preparations has shown that COX does indeed contain more than two Cu atoms, actually up to two and a half or three [671. While this finding per se i:, not entirely new, what is new is the fact that this third Cu (or one-half Cu, is now taken serious, whereas it was considered a contaminant in earlier years. And this third Cu is not EPR detectable; thus it would have the properties of the Cu required to give the mixed valence pair, as it seems to exist in N,O reductase. However. if’ there is only one-half Cu per COX monomer, this would not do. Furthermore. in more recent work [68] it was found that on monomerization the extra copper is actually


SYSTEMS:

MANZIANA

REPORT

187

lost. There are further problems with invoking a third Cu, as will become apparent in a presentation by 3. A. Fee, given on the following day. There is a bacterial COX that shows the EPR signal typical of Cu,, but does not contain more than two Cu atoms per molecule. That Cu, could be the second Cu in the mixed valence pair is quite unthinkable in view of all that is known today. I would rather say that more examples of two Cu COXs should be sought and those found thoroughly examined for a third Cu. As is well known to all those experienced in metal stoichiometries, the accuracy of the molecular weight determination of the protein is as important in such cases as that of the metal analysis. The further development of this problem is awaited with some suspense; it would not be the first time that a longstanding problem, as that of the nature of Cu, in COX, finds a solution from work that initially seemed to have no relationship to it whatsoever. After Kroneck’s lecture Dooley presented absorption and MCD spectra (taken at 4.2 K) of N,O reductase compared to those of COX which showed great similarities in the region attributable to cu.

HEMOCYANIN The next session was devoted to hemocyanin (Hc). As Francesco Ghiretti pointed out in his introductory remarks, this was the first time that, at a Manziana meeting, a whole session was devoted entirely to this subject. Understandably, even in this, at the molecular level rather intractable field, the advances in protein chemistry, molecular biology, genetics, and crystallography have brought many new insights, as became apparent in Anna Ghiretti Magaldi’s broad overview: “Hemocyanins, structure, amino acid and gene sequences, evolution,” which followed the introduction. She paid particular attention to the vexing question as to the relationship between molluscan and arthropodan Hc. She reported that at a recent meeting the suggestion was made that molluscan and arthropodan Hcs have evolved independently from a common ancestral mononuclear Cu protein, namely the copper B polypeptide of Hc. Ninety percent of the sequences of the two Hcs are not homologous, the two types of Hcs have very different structures and represent two different classes of proteins: they are products of convergent evolution. It has also been proposed that both Hc and tyrosinases are derived from an ancestral mononuclear CUB polypeptide. In the regions containing his residues, which are involved in metal coordination, homology in amino acid sequences of Hc and tyrosinase have been found [69]. In some respects the molluscan Hcs are more similar to tyrosinase than they are to the arthropodan Hcs (70). A comparison of the gene sequence for tarantula Hc subunite with the sequence encoding for mouse tyrosinase revealed great differences [71]. Molluscan Hcs however, are related to tyrosinases rather than arthropodan Hcs, suggesting that arthropodan Hcs and molluscan Hcs have most likely evolved from different ancestors. On the other hand, DNA sequence analysis for two insect larval serum proteins indicates a possible relationship with the Hc gene [72, 731. Analogies to these proteins are also found in the primary and quaternary structures and by crossreactions with monoclonal antibodies. In the subsequent talk B. Salvato presented a comprehensive summary of the principal reactions, which have been assigned to the various forms of Hc-naturally occurring and those produced in the laboratory.

188

H. Beinert

APO

--$c

SCHEME occurring

4.

F-hAOIl

Summary

or produced

having the “‘fast” loaded protein

of

the

principal

rcactmn
t’met

“i:!ircd

ant!

ialcnc.~.

the t’oilo~ ing ~~c*“I’osc’~p’c;‘li~

i FIN. 7;. 11IM! lx

dcrl\‘atr\.es

that gave the first +eL~tro~cqic

considering Karlin

Karlin

c.1~~ ft11,

ligand

When

and multiple

are

;_~ccnri~ Ascribed

mimL

‘I‘hc I)~T:u~

three tT\ the

morcty

i L:,quite ~i&?rcnt.

that for occurs.

Action.”

rmccf re\rrslbl>

rhc connciting

which

(Fig.

group

bctwscn

An inler’mi.~il,ltt:

in

inorc !riticn-

similar- :capable of proton translocation. While the sequence of the protein has not been determined. amino acid analyses show the presence of 1.O _t 0.1 cq’h and X.5 .t Cl.5 his residues [I I?‘]. Fee described an unusual reaction of cytochromc 00; with cyanide in which the cytochrome a3 autoreduces, cyanide binds to CI,, and binds weaki!, to Cuf3& EPR and ENDOR spectra of this species were presented which buggest that four histidine residues might be ligated to Cu, in a planar arrangement. If this is so, one can propose a model accounting for all the his residues in these proteins as liganda FO the metals: in ha,. two his bind Cu,, two his bind cytochrome b, one his binds to cytochrome Q~, and four his bind to Cu,. In an,, Cu, binds to the twc9 conserved his residues of subunit II while cytochrome u, Cu,, and cytochrome Q, bind the seven conserved residue> in subunit 1. thus accounting for all the conserved his


SYSTEMS:

MANZIANA

REPORT

195

residues in subunits I and II. If this ligand pattern is thought to hold also for mammalian COX, there would be only one cys available for any Cu site. This is the more disturbing, since the EPR signal of CuA is not significantly different in cytochrome ba, from that seen in mammalian COX. Fee then made an excursion into the molecular biology of the Thermus enzyme as reported in references 118 and 119. This presentation was followed by M. Brunori, who dealt with the classical COX and some of the yet unanswered problems concerning electron transfer to and within the oxidase. The first question he asked was that of the primary e- accepting site: is it Cu, or cytochrome a? Brunori made use of COX modified by pHMB treatment, which largely, but not entirely, eliminates Cu,. With this preparation he studied the burst kinetics following the addition of c by stopped flow spectrophotometry. The conclusion was that a is competent in accepting electrons from cytochrome c and, conservatively, it can be stated, that Cu, certainly is not the only entry site for electrons from c [ 1201. The conservative attitude stems from some remaining uncertainties in this type of experiment, because not all the Cu, becomes modified and there, unavoidably, is some other damage to the enzyme in the procedure. Brunori reminded the audience of work done by Yamanaka on COX from Cu deficient Nitrosomonas europaea [121]; which contains only about 10% of the Cu, found in the enzyme from normal cells, but still shows about 70% of the activity with horse heart cytochrome c and an unchanged KM. However with cyt c 552 from Nitrosomonas the activity is only 27 % with a 3-fold lower K,. Nevertheless these results point in the same direction as those of Brunori, namely that Cu, cannot be the only entry site of electrons into COX. The second question posed by Brunori was, whether a covalent complex of cyt c with the enzyme is relevant to e- transfer. Stoichiometric complexes of the enzyme were prepared with the use of dicyclohexylcarbodiimide with cyt c containing Fe, as normally, and cyt c containing Zn. The results of transient and steady-state spectroscopies were that covalently bound cyt c does not transfer electrons efficiently to a, whereas externally added cyt c is active in em transfer. The Zn containing complex showed the same behavior when bound to the cyt c binding site. Brunori concludes from these experiments that the classical stoichiometric binding site is not competent in e- transfer. In the consideration of the experiments reported by Brunori the rate of electron exchange between a and Cu, remains an important point. As Brunori pointed out in closing, work carried out in two different laboratories by different approaches [122, 1231 showed that this rate is about 100 times faster than had been concluded from previous experiments, so that it would never be rate limiting in experiments of the kind described. At this point, F. Malatesta took over and reported new experiments on the rate of electron transfer from a and Cu, to the binuclear center in COX. The rate of reduction of a3 was followed by transient kinetics in the presence of CO under a number of different conditions. The internal e- transfer rate accounted for the turnover number, with both displaying the same pH and temperature dependence. Comparison of the time course of cyt c oxidation and cyt a3 reduction indicates that two electrons are transferred internally and with different rates to the 0, binding site. Shown below is a scheme accounting for these observations [1241. Note that the e- transfer to the binuclear site is rate limiting; that e- move one at a time either sequentially or through independent e- transfer pathways; that the central intermediate with one e- in the binuclear site does not bind CO. This work makes a

196

H. Reinerr

.

BLUE

COPPER PROTEINS The first session of the last full meeting-day

started with a lecture

in which

he gave

the audience

some

highlights

G-proteins

by

impressive

range

of

biological mutagenesis

an

approaches.

As

an example,

studies on Azrzrin

(AZ)

of a program

contemporary Canters

hiophysical

mentioned

of Pseudomowcr~

by G. $V. Canters. clt’ investigation.\ and

his group’:,

~cw,~,~Kw.

on

molccuiar NMR

hv ‘ii:hidi

and it w3s


SYSTEMS:

MANZIANA

REPORT

197

shown that there is no evidence for the existence of redox-inactive forms, as had appeared from previous work [126, 1271. Electrons reach and leave the Cu through Cu-ligand his 117, both in the e- self-exchange reaction and in the reaction of AZ with its presumed physiological partners, cytochrome cs5,, and nitrite reductase. His 117 pierces the hydrophobic patch in the protein surface and is accessible from the outside [128, 1291. The state of protonation of his 35 affects the redox potential of the Cu, and the oxidation state of Cu affects the pK, of his 35; this together with the slow interconversion of the his 35 species is sufficient to cause the observed slow phase in the AZ to cytochrome c reaction kinetics. Canters then turned to Amicyanin from Thiobacillus versutus, the blue Cu protein in the methylamine oxidizing redox chain of T. versutus [ 130, 13 I]. Its 600 MHz ’ H NMR spectrum has been assigned by means of 2D NMR. The secondary structure has been established and determination of its 3D-structure is underway. The secondary structure resembles that of plastocyanin, but the peptide (21 residues) preceding strand A seems to contribute an extra strand to sheet I. Amicyanin becomes redox inactive at low pH. The inactivation is caused by the dissociation of ligand his 96 from the Cu when the protein is in the reduced form. A thermally activated transition from reduced to oxidized protein at low pH is thought to be possible only when electron transfer is accompanied by proton transfer, resulting in a high activation barrier for electron transfer. Canters then considered the effect of mutations on spectroscopic and redox properties of blue copper sites. Mutations of type-l Cu proteins, even when applied outside the first coordination shell of the Cu, produce small but distinct variations in optical properties (600 nm band), EPR parameters, and redox potential. A detailed theoretical interpretation of the effects requires precise structural information about the Cu site (132). Some of the observed changes appear attributable to charge effects and provide a test for dielectric medium theories of proteins. An additional aspect of enzymology of great present interest is immobilization of enzymes on a solid support. Canters mentioned two approaches pursued at present in his laboratory. First, the signal peptide can be mutated so as to prevent processing of the protein. This provides for an N-terminal tail which can be positively charged to enhance Coulomb interaction with negatively charged surfaces. Second, a ligand can be removed, creating a hole in the protein envelope through which the Cu becomes accessible for external ligands; the external ligand may be anchored on the surface. The latter approach has been followed, for instance, with the construction of a mutant (H117G) in which a Cu ligand (his 117) was replaced by a glycine. The mutant appears to still bind Cu, which, however, gives rise to a type-2 EPR spectrum and an unusual absorption band around 420 nm. Subsequent addition of imidazole restores the full type-l characteristics (type-l EPR spectrum, 628 nm absorption) of the azurin. Finally, Canters reported on his ongoing work on the methylamine dehydrogenating (MADH) redox chain and its components [133]. According to a simplified picture, the methylamine oxidizing redox chain from T. versutus consists of MADH, amicyanin, cytochrome cssO, and a terminal oxidase. Until recently MADH was thought to contain PQQ as the prosthetic group, its precursors presumably being a glu (57) and an arg (108). The larger part of this chain has now been cloned. One of the surprising findings is the occurrence of trps at positions 57 and 108 in MADH (not glu and arg as guessed previously), which raises questions about the structure of the cofactor. The broad ranging work of this group on Cu proteins must be followed with interest and expectations.

198

H. Beinert

The next speaker was S. S. Hasnain, who discussed the complementary nature of EXAFS and crystallographic methods for studying structure-function relationships in blue Cu proteins. He pointed out that details such as the local environments of metal atoms in metalloproteins are not as precisely defined as would be expected from. say, a “small molecule”’ x-ray structure analysis. Such detail is essential for understanding the chemistry of these reaction centers. In contrast u ith EXAFS, only the local environment of the metal site can be investigated because of the short mean free path of the x-ray generated photoelectron whose scattering gives rise to the structure of the EXAFS data. This localized nature of the phenomenon gives 3. major advantage in that local structure of the metal site can be probed with an accuracy’ often approaching that routinely achieved in small molecuie crystailography An additional advantage is that EXAFS data can be collected on both queous and Thus. m addition to borate crystalline proteins with very similar resolution. definition of the metal site in the native protein, it is possible to study changes in the local chemistry upon a biochemical reaction. These changes are often quite subtle and within the error limits of a crystallographic determination. Hasnam emphasired that, although crystal structures of a number of Cu proteins are now ;i:ailahle, it is desirable to study these proteins under a variety of conditions in order to dixccrn the structural features which, e.g.. are responsible for thz wide range of evidenti> tunable redox potentials which can arise from the same basic strttctura! framework. Hasnain tnentioned that he and a number of colleagues have started arc international program in which it is intended to determine the crystal structures of certain selected proteins at high resolution and also to improve the definition of the metal site by the use of EXAFS. The EXAFS studies are to he done under a \arietv t)f conditions. e.g., of oxidation state or pH. Hasnain then mentioned some details of fitting procedures and illustrated these with the examples of plastocyanin anti rusticyanin. The first shell analysis & the EXAFS data for rusticyanin cleari~ shows that II cannot bc simulated by the first shell of nitrogen atoms of the histidine at LLI. 1.96-2.02 A and a sulfur from cysteine at ca. 2.14--I!. 17 A. A further strong contribution from a sulfur atom. presumably from methionine at ca. 2.5--2.6 .& is also essential. These studies have shown that the rhomhrc nature of the EPR and the 450 nm band in the optical spectra appear when the fourth copper iipand makes a close approach. Cu-X, .- 2.6 ,&. Finally. Hasnain turned to an investigation of four mutants of P. wruginosa Al which were produced to obtain information on the role of met 12 1. a copper ligand thought to play a crucial role in spectrochemical as welt as rtdox properties of these proteins. All of the mutants, including the one where the protein is terminated at residue 120, display characteristic properties of blue copper centers. demonstrating that the methioninc sulfur is not essential for creating a blue copper site. In the next lecture. Sakurai reported that direct electrochemistry of three blue copper proteins. Thus wrnicifera stellacyanin. Ctrcut~zis .sativr4splantacyanin, and Achromohacter cycioclastes 1AM 1013 pseudoazurin has been achieved at a glassy carbon electrode over the range of pH 4- 11 in the absence ot mediators an d promoters [ 1341. The formal potentials E”’ for stellacyanin, plantacyanin, pseudoazurin are 0.18. 0.32, and 0.28 V ivs NHE), respectively, at pH 6.0. Thcsc values are identical with those previously determined by conventional potentiomrtric titrations. The three blue copper proteins behave as effectively symmetrical redox systems over a wide pH range. However. cathodic and anodic wave peak Potentials iUld


SYSTEMS:

MANZIANA

REPORT

199

of the three blue copper proteins show diverse pH dependencies, suggesting that the electrons might be passed to and from the copper center via different pathways. In addition, the slopes of the shifts of the anodic and cathodic waves, which are much smaller than - 59 mV at pH < 10, indicate that the electron transfer processes between the electrode and the three blue copper proteins are not accompanied by H+ transfer. The heterogeneous rate constants (k’) for stellacyanin, plantacyanin and pseudoazurin are 3 x 10-4, 4 x 10m4, 2 x lop3 cm/set, respectively. The oxidation-reduction processes are almost reversible or quasi-reversible at pH < 10 as indicated by fairly narrow cathodic and anodic peak separations (A Ep = 60-90 mV). At pH > 10, A E, values begin to increase because of a protein structure change, which is partly reflected also in the absorption and ESR spectra of the proteins in the oxidized form. Joint work of 0. Farver and I. Pecht was reported in the next two lectures, the first given by Farver and the second by Pecht. They have investigated parameters controlling long-range intramolecular electron transfer processes in proteins. The systems chosen were a) the blue single-copper proteins, stellacyanin (St) and azurin (AZ) and b) the blue multisite copper oxidases, lactase and ascorbate oxidase. Ru-modified stellacyanin was produced by binding Ru(NH&+ to solvent-exposed histidine residues on the protein. By pulse radiolysis a Ru(II)-St[Cu(II)] species was produced, and the intramolecular e- transfer was studied over a temperature range from 2.5 to 40°C. The rate constant at 25°C is 0.07 M-’ s-l; AH* = 19 kJ mol-‘; AS = - 201 J K- ’ mall ‘. The slow rate is most likely due to the relatively long etransfer distance (1.6 nm through-space) combined with a small driving force (10 kJ mall’) [135, 1361. The single disulfide group linking cysteines 3 and 26 on AZ can be reduced pulse radiolytically by CO, radical anions. The disulfide radical, RSSR- thus produced decays by an intramolecular process, transferring an electron to the Cu(II) center which is at a distance of 2.0 nm. The reaction was studied over a temperature range from 2 to 45”C, for several different azurins (Table 1). The activation parameters are characterized by relatively small activation enthalpies and large negative activation entropies. The latter are due to the large distance separating e- donors and acceptors, making the reaction highly nonadiabatic. Electron transfer is most probably mediated by coupling of electron donor and acceptor through a combination of covalent and hydrogen bonding, as well as non-covalent contacts, and the pathway may depend on very specific interactions within each protein. Using the predictive theoretical method for electron tunneling

TABLE 1. Kinetic and Thermodynamic

Data for Electron Transfer from a Pulse-Radiolytically Generated Disulfide Radical to Type-l (Cu(I1)) in Four Different Azurins Azurin P. ser. A. spp. P. fluor. A. faec.

k, s-’ 44+7 8.5 + 1.5 22 + 3 11+2

AH*, kJ mol-’ 44.5 k 4.0 16.7 + 1.5 36.2 + 2.9 54.4 rt 4.8

AS, J K-’ - 56.5 + - 171 k - 36.2 + -45.0 +

mol-’

E’, mV

7.0 18 2.9 5.2

304 260 266

P. aer: Pseudomonas aeruginosa; A. spp: Alcaligenes species; P. fluor: Pseudomonas fluorescens; A. faec: Alcaligenes faecalis.

200

N. Reiner:


SYSTEMS:

MANZIANA

REPORT

201

be bound, including diatomic and monoatomic intermediates or reduction products. McMillin felt that the flexibility in the protein, as demonstrated above in the temperature studies, and the cooperativity between the type-2 and type-3 sites, as observed in the anion binding studies, would eventually furnish clues for understanding how the enzyme can so efficiently carry out the reduction of 0, to H,O. In the next lecture Maria Theresa Graziani spoke about attempts to selectively reduce type-3 Cu inRhus vernicifera lactase by thiol reagents. Chelating agents of different efficiency were tested on lactase to investigate the properties of its three different types of Cu ions. Thus far only type-2 Cu could be selectively and reversibly removed from the protein under reducing conditions, namely by the chelating agents nitrilotriacetate [ 1441 (NTA), N,N-diethyldithiocarbamate (DDC) [ 1451, and dimethylglyoxime (DMG) [ 1461, with the order of efficiency being NTA > DDC = DMG. EDTA was unreactive by itself, but had an effect when used in combination with the two last-mentioned chelators. Dimercaptosuccinic acid (DMSA), under anaerobic conditions and in the presence of excess ferrocyanide as reducing agent, leads to removal of type-2 Cu as does NTA. From the inactive T2D derivative, 70% of the original acitivity can be recovered, when it is reconstituted with CuGSH [147]. When mercaptoethanol or dithiothreitol is present in addition to DMSA, a protein is obtained that shows modified spectroscopic properties. In this new species the 330 nm band is absent, the absorbance in the 650-800 nm region is decreased, while the EPR spectrum shows a modified type-2 signal with a narrowed hyperfine splitting, viz. A = 12.0 mT, while for the native protein A = 18.4 mT. The DMSA-treated lactase retains 30% of the original enzymatic activity and 80% of the original Cu content, with an equal distribution between para- and diamagnetic ions. On addition of H,O, most of the 330 and 650-800 nm absorption is restored, indicating reoxidation of type-3 Cu which had been reduced by the DMSAmercaptoethanol treatment. The properties of the new species (before reoxidation!) are similar to those of the form of lactase containing reduced type-3 Cu [148] previously detected in native lactase. Graziani considers the possibility that a disulfide bridge close to the trinuclear Cu cluster [cf. 621 is affected. As the last feature of this session M. A. Mateescu took the audience on an excursion into biotechnology, when he presented his paper on the immobilization and activation of lactase on agarose columns. He recently introduced a new type of ready-to-use p-benzoquinone (p-BQ)-activated support (agarose) for one step biochemical immobilization (yield lo-90%) [149] by modifying a classic procedure (activation followed by immobilization) of Brandt et al. [ 1501. A peculiar behavior was observed for lactase immobilization inasmuch as the enzyme retained specific activities of 150%) i.e., a higher specific activity of the enzyme after immobilization. The result was correlated with the presence of hydroquinone (HQ)-groups (byproduct of the activation)-a substrate analogue for which lactase exhibits a high affinity. Thus, the specific lactase immobilization probably occurs with a simultaneous affinity retention on the HQ-groups, while the p-BQ groups generate simultaneously the coupling so that the whole process can be considered an “affinity immobilization” [149, 1511 (Scheme 7). For the case described, Mateescu suggests [cf. 1521 that the specific activity of the immobilized lactase is due to the accumulation of the “active” form on the support which contains the reducing substrate (HQ). On coupling at pH 6 and in the presence of oxygen this may generate the preferential immobilization of the “active” form of the enzyme.

202

H. Beinert

SCHEME 7. to Mateescu.

ASCORBATE

Schematic drawing of synthesis of support and activation of lactase. according

OXIDASE

With high resolution crystal structures appearing in increasing numbers these days, the excitement that used to accompany these events in the past is tapering off. However, when the structure that becomes available is of a type of protein that has so far not been seen in three dimensions, it still commands our attention [95]. The 3D structure of Ascorbate Oxidase (AAO) from zucchini is indeed the first one reduction of 0, to water. A. known for an oxidase that catalyzes the 4-e Messerschmidt gave details of the 1.9 A structure of AAO. It consists of two identical subunits of 552 amino acid residues related by a diad axis. Each subunit has a globular shape and is built up by three domains arranged sequentially along the polypeptide chain and highly associated in space. The folding of all three domains is of a similar p barrel type. It is distantly related to that of the blue Cu proteins plastocyanin or azurin. No carbohydrate was found associated with the protein. Each subunit has four catalytic coppers bound as mononuclear and trinuclear species. respectively. The mononuclear Cu is a type-l Cu with bib. cys, his. met as ligands. It is located in the third domain. The trinuciear cluster has eight his ligands. It ma) be subdivided into a pair of Cu atoms with six his ligands arranged as a trigonal prism. and a single type-2 Cu. The pair represents the type-3 Cu. The remaining Cu (type-2) has two his ligands. There is evidence for two nonprotein !igands of the trinuclear center, namely an OH- bridging the Cu pair and a water or OH bound to the type-2 Cu. The protein ligands are supplied symmetricaIl> by domains 1 and 3. The type-l Cu is the binding site of the reducing substrate. and the trinuclear Cur cluster is the binding site for molecular 0,. The entry for the electrons is the type-l Cu. They are then transferred to the trinuclear Cu site which is able to store three electrons simultaneously. It is of great interest whether the intramolecular e transfer follows a through-space, a through-bond. or a mixed through-space. and through-bond mechanism. Candidates for parts of the e transfer pathway are the Cu-thiolate bond, the hydrogen bond from the carbonyl-oxygen of cys 509 to N, of his 508 and the a-electron system of the imidazole ring of his 508. ‘The question arises here whether a preferred e transfer is possible through a hydrogen bond. Messerschmidt proposed a model for the AA0 reaction. which assumes the binding of O2 as 1.2 p.-peroxo species at the fully reduced enzyme after release of the central OH ligand. (Fig. IO). It also includes an oxygen radical intermediate which has been detected by low temperature EPR during reoxidation studies of tree lactase


FULL”

FIGURE

10.

lED”EED

SYSTEMS:

EYL”“.?

Proposal for the catalytic mechanism

MANZIANA

Q

K)*s,aL”

WUYD

REPORT

As

1.x*-

cc”oxo

203

sPeCCn!*

of ascorbate oxidase.

[153]. It may be mentioned here that the nucleotide sequence of the full-length cDNA for AA0 from cultured pumpkin cells has become available since the meeting [154]. Appropriate to the discussion of type-2 Cu and the type-3 copper pair and their ligand interactions, P. A. Clark made some critical remarks emphasizing that one has to view reactions that any of these metal centers undergo in the light of the fact that one may be-and under most circumstances actually is-dealing with a system of coupled spins. Clark pointed out that the crystal structure of ascorbate oxidase [95] shows a region of electron density between the type-2 and type-3 copper ions that appears to be a water derived ligand. The presence of a potential endogenous bridging ligand between the type-2 copper and type-3 pair suggests that these ions may be magnetically coupled to form an S, = 3/2 cluster. For a trinuclear cluster of three spin = l/2 ions, with a strong antiferromagnetic exchange interaction between any pair of ions, the cluster will have a doublet ground state with a doublet and quartet excited states. This case is applicable to lactase where the type-3 copper ions are EPR silent. Magnetic susceptibility measurements were taken on met T2D lactase, were the type-2 copper of the cluster has been selectively removed, to probe the magnitude of the interaction between the type-3 copper ions. No deviation from Curie-law behavior, due to the paramagnetic type-l copper, was observed for met T2D lactase to 200 K, setting a lower limit for the exchange interaction between the type-3 pair at - 25 > 500 cm- ‘. Magnetic susceptibility measurements on TlHg

204

H. Beinert

lactase, where the type-i copper has been removed and replaced by Hg'+. also show linear Curie law behavior to 200 K with a magnetic moment which is consistent with a single S = l/2 ion or only population of doublet states of the trinuclear cluster. Magnetic susceptibility measurements are not useful for detection of excited states with the same spin, as they have the same magnetic moment. The magnetic model (Fig. 11) indicates that the wavefunction of’the excited sjtate doublet will not be localized but will contain contributions from all three copper ions. This raises the interesting prospect that new MCD features may bc observed when the excited doublet state becomes thermally populated [ 1021. Variable temperature MCD spectroscopy is currentlq being employed to look for thermal population of an excited doublet state in the trinuclear cluster of the multicoppcj- oxidase. laccasc. A. Marchesini then reported on the reduction of .4AO b> ilavin rcmiquinones. which was studied using laser flash photolysis under anaerobic conditions. ‘T!pe-I Cu was reduced rapidly in a reaction dependent on protein r:ancentration with k = 2.7 x 10 M ’ s ‘. This reduction was followed by a rapid rntramolecular reoxidation of approximately 50%’ of the reduced type-l 6% \i,ith L = 160 s ‘. Removal of type-2 Cu slightly increased the rate of reduction of type-i Cu. frut did not alter the rate constant for type-l Cu reoxidation. Bindin, 0 i>t’i’yanide eliminated the reoxidation of type-l Cu in a concentration dependent rrranner. wtthout altering the kinetics, both in the native and type-2 depleted enzyme. “hcsc re~lts indicate that type-3 Cu is the immediate e acceptor f’rom type 1 Cu snc! that cyanide binds to the type-3 site. Addition of HzO, increased the extent of rcoxidation of type- I Ca without affecting the kinetics, This is most likely due to the fact that in the native enzyme type-3 Cu is not fully in the osidizcd state. Miilimol;i: ionccntrations of fluoride and azide were without efl’eet on the kinetics of t>pe-E C’u reduction :md reoxidation. In the lecture following. Luciana Avigliano was concerned with a variety of aspects of AAO. such as distribution, localization, possible phvsiologlcal flmction. photochemistry, and carbohydrate content. Antibodies raised against homogeneous AA0 were used for the tmmunohistochemical localization oi thr rnz~mc in green zucchini iCucurbita peym medullosa). AA0 was found in ail the specimens examined, namely in vegetative and reproductive organs. At the cellular ieke the enzyme is associated with the cell wall and the cytoplasm [ 1551. With cuiturcs ot green zucchini callus, addition of Cu as CuSO, increaycd AA0 activity substantially. an effect counteracted by abscisic acid. An equivalent increase is not found in mRIiA assays. Avigliano also reported that with deglycation of the protein th? activltq ot AA0 goes up. The three dimensional x-ray structure oi‘ green Tucchini AA0 suggests a better exposure of the possible ascorbatc binding site [ 156j. On photoouldation the activity decreased logarithmically with exposure time: addition ol mannito1 provided no protectior::. The apoenzyme ~.a’; found to hc more \enkitivc than the


SYSTEMS:

MANZIANA

holoenzyme. Similarly, in experiments on heat sensitivity T2D-enzyme, carried out by DSC, the apoenzyme showed 20°C lower than both the holo- and T2D enzymes (35).

REPORT

205

of holo-, apo-, and a melting point some

CERULOPLASMIN G. Musci referred to recent work [157] in which it had been shown that ceruloplasmins (CP) prepared by a new procedure do not have the usual EPR spectrum in that the signal of type-2 Cu is absent. Studies on the redox behavior of the protein, which had been anaerobically reduced by ascorbate and subsequently exposed to air, then revealed that a transient intermediate with type-2 Cu features appears in the EPR spectrum after reduction of the chromophore at 330 nm. The original optical and EPR spectra reappeared within a few minutes, which is at variance with the observations on classical preparations of CP, where only 50% of the blue color can be restored by 0,. The conclusion was that in oxidized CP the type-2 Cu interacts with the type-3 Cu such that its EPR signal becomes undetectable, but that this interaction can be eliminated by reduction of the type-3 pair. The EPR spectrum of the form of CP obtained by Musci et al. corresponds to 2.5 (out of a total of 5) paramagnetic Cu atoms per molecule and has been simulated for turtle protein [158] by considering the presence of two non-equivalent type-l Cu atoms and a contribution of a broad resonance at g = 2 which accounts for approximately 0.4 spins per molecule. Musci and his colleagues have also reinvestigated the reaction of NO with CP. Treatment of chicken CP with NO bleaches the EPR spectrum of type-l Cu, which is, however, restored by removing the gas. There is no type-2 Cu signal in the EPR spectrum of NO-treated CP, at variance with previous reports [159] on the mammalian proteins. However, when the type-2 Cu signal is present in the spectrum of the protein as isolated, e.g., in samples subjected to prolonged storage or prepared by classical procedures, it persists with unchanged intensity after addition of NO. However, the type-2 transient intermediate observed during reduction by ascorbate is completely bleached by NO and does not reappear on removal of NO. The optical spectra of oxidized and partially and fully reduced chicken CP treated with NO reveal that after addition of the gas the absorption at 600 nm becomes stabilized at around 50% of the intensity of the native oxidized protein, irrespective of the initial redox state of the blue Cu ions. The modifications at 600 nm are fully reversed by removing NO and adding 0,. However, new absorption bands appear below 500 nm on incubation with NO. These bands do not disappear after removal of NO or after prolonged dialysis. The redox behavior of CP is modified by NO, in that the protein shortly exposed to NO is reduced by ascorbate 3 to 5 times faster than the corresponding untreated control. While the interaction of NO with type-l Cu can be rationalized by assuming reversible formation of a diamagnetic charge transfer complex between the blue Cu ions and NO itself or a group on the protein reduced by NO, the irreversible formation of new bands between 300 and 500 nm and the modified redox behavior of the NO treated protein may indicate a tight binding of NO (or other nitrogen oxide) to the trinuclear cluster. In the discussion following this lecture Lilia Calabrese emphasized that the absence of the EPR signal for type-2 Cu in the new type of CP preparations, as introduced in Musci’s talk, does not mean type-2 Cu is absent, neither does the appearance of this signal after aging of the enzyme mean that type-2 Cu is arising by degradation of other Cu centers present, but that all these phenomena are related to changes in the interaction of the type-3

206

H. Beinert

pair and the type-2 Cu. The most obvious of these changes would be reduction of type-3 cu. In the next lecture Maria Carmela Bonaccorsi di Patti considered the multidomain structure of CP as deduced from studies carried out by calorimetry in connection with limited proteolysis 1351. The CPs from sheep, chickens, and turtles showed a similar structural organization in three calorimetric domains, but the temperature of unfolding varied from 57.8”C to 71.2”C and 82.1 “C from turtle to sheep and chicken. The spectroscopic and catalytic properties were completely lost at temperatures corresponding to the unfolding of the least thermostable domain for sheep and chicken CPs and to the unfolding of the most thermostable domain in turtle CP. Turtle CP was insensitive to proteolysis by plasmin or trypsin, whereas sheep and chicken CP gave rise to fragments which did not dissociate under non-denaturing conditions. With plasmin the extent of degradation was very limited and the overall thermal stability was essentially unaffected in both proteins. With trypsin, cleavage was much more extensive and resulted in a significant decrease in thermal stability. Cu removal caused the rearrangement of the molecule into only two calorimetric domains. suggesting a role of the metal atoms in organizing a new calorimetric domain, which was tentatively assigned to the least thermostable cooperative unit of the native protein. This part of the protein appears to be the primary site where modifications induced by different perturbations, such as proteolytlc attack or aging selectively occur, which results in a rearrangement of the whole molecule. as recognized by altered magnetic and calorimetric properties

METALLOTHIONEIN Next, Valeria Culotta gave the audience a lively excursion into molecular biology with her discussion as to how the expression of the MT gene is regulated, but not before confessing to her audience that, notwithstanding her name, she was not a native of our beautiful host country, but another Yankee from Baltimore, albeit from Johns Hopkins. The MT gene of S. cerevisiae is transcriptionally regulated by the combination of Cu ions and the ACE1 regulatory factor. ACE1 exists naturally as an extended apoprotein incapable of binding DNA. However. in the presence of Cu the protein coordinates the metal, thereby changing its conformation to that of a DNA-binding metalloprotein 11601. This conformational switch 1s specific for Cu(1) and Ag(Ij ions, and occurs in a highly cooperative fashion. The cell can therefore respond to a small change in Cu concentration by a large change in MT gene expression [ 1611. Cu-ACE1 binds specifically to MT promoter sequences and facilitates the assembly of an active transcription complex involving factors of the general polymerase II transcription machinery 1162, 1631. A combination of biochemical, genetic, and biophysical analyses infer that activated Cu-ACEI contains a polynuclear Cu(I)-S cluster, or a “Cu fist” structure that is remarkably similar to the Cu cluster of MT itself. Culotta, therefore, speculated that ACE1 may have evolved by the addition of DNA binding residues to a primordial metal binding structure. The Cu fist contains 4 to 8 Cu or Ag per molecule and x-ray absorption spectroscopy has shown that the metal environment is trigonal. The Cu-S distance is 2.24 .4. Weser added to this by remarking that two of the Cu atoms are easily removed from the protein by chelators and that the whole ACE1 protein has a molecular mass of 77 kD and the Cu fist of 1.5 kD, The next presentation, by I. Bertini, did not actually deal with a &-protein. rather


SYSTEMS:

MANZIANA

REPORT

207

with a Co-substituted Zn/Cd protein, Metallothionein (MT). The lecture derived its justification for being on the program from the fact that copper-thioneins also exist, and that Co has been used successfully as a substitute for Zn as well as for Cu in some proteins, such as, e.g., in SOD. The virtues of Co in this respect are its paramagnetism and preference for tetrahedral coordination as found in, e.g., blue Cu proteins and in a number of Zn proteins. While, generally, the distinct optical properties and EPR spectra of Co have been exploited, Bertini and his colleagues have found that the high spin (S = 3/2) and the spin relaxation properties of Co(I1) are particularly favorable for observing relatively sharp ’ H NMR lines of paramagnetically shifted protons. The 3D structure of MT is known from x-ray diffraction as well as from NMR spectroscopy. There are two domains to the molecule, each housing one tetrahedrally coordinated Me-thiolate cluster, the A-cluster with 3 Me and 9 cys residues and the B-cluster with 4 Me and 11 cys residues (Fig. 12). Zn/Cd MT has been extensively studied by ‘13Cd NMR in forms of various Zn/Cd ratios and the Co(I1) derivative has been prepared and its electronic and EPR spectra have been investigated. Now, Bertini and his colleagues have undertaken to do ’ H NMR on the all-Co(I1) form, (Co(II)7). When the titration of apo-MT with Co was followed by ‘H NMR, the spectra were poorly resolved until about 5 moles of Co per mole of protein were taken up, but spectra improved dramatically on further addition of Co, so that sharp and well separated signals could be observed; more than 20 isotropically shifted signals were spread between 300 and 100 ppm. The signals were assigned to the Co,S,, cluster domain (Fig. 12), by comparing the spectra of the Co,MT and the Co,Cd,MT derivatives; the Co, cluster probably undergoes dynamic phenomena that cause broadening beyond detection of the 1H NMR lines of the coordinated cysteines. Most of the far isotropically shifted ‘H NMR signals are believed to arise from the @CH, protons of the 11 cysteines involved in metal coordination, experiencing large contact contributions [ 1641. Titrations were also followed by measurements of magnetic susceptibility. With up to three moles of Co the susceptibility rose as expected, but then dropped on further addition up to four moles, and again resumed a rising pattern after this. The interpretation of these observations is that initially, Me-ions randomly occupy non-adjacent sites with preference for the B-site; however, between the addition of three and five Me-ions the B-cluster becomes organized and the Me-ions occupy vicinal positions, such that Me-Me interaction becomes established through the thiolate ligands. Consequently, paramagnetism is lowered through spin-coupling, apparently establishing the state, which provides the favorable conditions for a remarkably well resolved ‘H NMR spectrum. Bertini and his colleagues have proposed a theoretical model to describe the antiferromagnetic coupling operative inside the four metal cluster of Co,MT. The model permits one to calculate the electronic levels of the cluster and to predict the temperature dependence of the isotropic shifts of protons sensing the different

B

FIGURE MT.

12.

Schematic drawing of the Me,S,,

(B)

cluster of

208

H. Beinert

Me-ions. They could show that a set of five large and one essentially zero J value for the Me-Me magnetic coupling interactions can satisfactorily explain the experimental spreading of the isotropic shifts and their temperature dependence 11651. More recently, ‘H NOE experiments on Co,MT have allowed Bertini and his colleagues to proceed with the assignment of the ‘H NMR spectrum of the Co,S,, cluster. ‘H NOE experiments permit the detection of the proximal spatial connectivities existing among the isotropically shifted signals, assigned to the &CH, groups of the metal coordinated cysteines, with an upper limit of about 3.0 &. Geminal oonnectivities between the protons of the same methylene group were identified with high contidence for at least 9 out of the 1 I cys of the 4-Me cluster: iurther conncctivities were found between protons ot different cys B-CHr groups as well as between far and near isotropically shifted signals. Some of the latter signals were tentatively assigned IO the corresponding cys tu-CH protons. With the help of computer graphics analysis of the four-metal cluster, based on the structure in solution previously obtained for Cd,MT through 20 NMR, it was possible to proptosc tentative individual assignments of a few ‘11 NMR signais. In particular. Bertini proposed scqutncc specificassignments for the six methylene protons. whoss shifts exhibit xn anti-Curie temperature dependence, The assignment relies on the predictions from the thcoretical model [ 166j.

SUPEROXIDE

DISMUTASE

The last day’s session was mainly devoted to aspects of superoxide dismutase (SOD). M. Bolognesi lead off with a report on the status of the crystal structure determination of yeast CuZnSOD, which has been extended to 2.S ,& resolution [ 1671. The structure is homologous to that of bovine SOD and could. therefore. be solved by molecular replacement methods. The yeast enzyme crystallizer as a tetramer as opposed to bovine SOD which forms a dimer. Fifty--five percent of the amino acid residues are identical. Solvent molecules arc not yet included in the structure. This presentation was followed by a discussion by .A, LQsideri on the electrostat its of SOD, which have received particular attention in the past because of the possible relevance to substrate-steering toward the active cite. Drawing on six examples of SODS from various species, Desideri found that a similar electrostatic potential exists in all of these enzymes 1168, 1691. namely a generally negative potential, except at the entrance to the active site. Fee raised the question \)t‘ the selectivity of such an effect, as, for instance: Why are butfer anions not similarly accumulated at this site? The difficulties of coming to definitive ctnrsiuaions in this arca are ably highlighted in a. paper which appeared after the meeting and the interested reader may find there much information and related lircraturt. on thik topic ]17fll The meeting then turned to more biological aspects of SOD biosynthesis and function. 6. Rotilio spoke on the regulation of CuZn SOD in differentiating human K562 leukemia cells. it had been reported that CuZn SOD activity increases during erythroleukemia cell differentiation [ 171]. In addition. the human erythroleukemia cell line K562 has also been shown to bind ceruloplasmin to the celi surface and to internalize Cu from external ceruloplasmin: both processes are dramatically cnhanced during differentiation [i 721. Recently Rotilio’a group found that Cu(I)glutathione (GSH) can reconstitute Cu-free SOD with higher efficiency than any’


SYSTEMS:

MANZIANA

REPORT

209

other Cu-complex tested [ 1731. They suggested that this complex may also be a good candidate for the regulation of Cu-transport in vivo. The K562 model was, therefore, re-examined with special attention to the relationship between Cu-content and CuZn SOD activity. They found that a Cu-depleted SOD protein is present in K562 cells. However, differentiation of K562 cells does not imply induction or repression of the CuZn SOD gene; rather, the increase in the concentration of active enzyme is regulated post-translationally by differential delivery of Cu to a constantly expressed protein as a function of differentiation stage. The concentration of intracellular GSH, which increases during differentiation of K562 cells, is apparently related to the activity of CuZn SOD [ 1741. U. Weser then discussed the relationship between tissue Cu and the formation or elimination of oxygen-derived free radical species. The rise of serum-Cu-up to 300% above normal-is a well-known phenomenon in systemic inflammation. The cause of this phenomenon is not known. Fast proliferating cells, including melanoma, leukocytes, enterocytes, and yeast cells, form marked quantities of Cumetallothionein (MT) at the same time. Weser asked the question as to the significance of this Cu-thionein. He produced Cu-thionein experimentally in the course of intestinal Cu-transport and characterized it by chemical, physicochemical and immunological methods [175- 1771. Cleavage of this MT, e.g., by alkylation with iodoacetate and oxidation, gives rise to biochemically active Cu(I1) [178]. Oxidative depolymerization of hyaluronic acid, as induced by activated leukocytes [ 1791 can be inhibited by added Cu(I1) sulfate as well as by Cu-thionein. During cell activation oxygen free-radicals are formed in an uncontrolled manner. However, in activated polymorphnucleocytes, leukocytes, or macrophages, these oxygen species are dealt with by the reaction with Cu-thionein [ 1801. For example, in the presence of Cu(1) the formation of OH. or other transient oxygen species as well as the catalysis of superoxide dismutation in the presence of Cu(I1) must be considered; Cu-thionein appears to be an ubiquitously occurring active scavenger of oxygen radicals. In addition to the reactivity of Cu(I), the scavenging ability of the many thiolate sulfurs present in thionein is also of importance in this respect. Continuing along lines followed previously by Rotilio, Maria Rosa Ciriolo spoke on the regulation of CuZn SOD in yeast. This protein appears to be particularly sensitive to extremes of environmental Cu concentration. For instance, the amount of measurable intracellular activity of SOD increases as the Cu concentration in the medium is raised above 100 PM. It has been thought that this increase is due to the capability of Cu to promote the generation of intracellular reduced oxygen species such as 0;) H,O, and OH and that CuZn SOD might be required to counteract these events. Ciriolo has investigated the effect of various experimental conditions on CuZn SOD in Saccharomyces cerevisiae (D273-10B). Yeast cells were grown under normal aerobic conditions, under anaerobic conditions, and supplemented with 9% glucose, and also under these three conditions with addition of Cu. The results of determinations of CuZn SOD-mRNA, immunoreactive CuZn SOD, CuZn SOD activity and cellular Cu content suggest that the delivery mechanism of Cu ions to the protein plays a major role in the regulation of CuZn SOD by oxygen. Furthermore, synthesis of the protein seems to be directly induced by Cu in an oxygen independent fashion at the transcriptional and post-transcriptional levels [ 1811. These results suggested the possibility that CuZn SOD is coregulated with Cu MT via a transcription activating factor, such as, e.g., ACEl, the protein responsible for activation of CUP1 expression. The following experiments were therefore carried out: DTY26, a

210

H. Beinert

A! UASd

UASc ..___._____--_-~----.

-177 -234 -217 -197 TTTCTTCTAGAAGCAAA......GCTGAACCGTTCCAGCAAM..

MT !coding)

TTACTGGAAGTACAGAA......GTTAAACCGGTGTGTCGGAA... :225 -209 -170 -150

33 bp

.TATA box

48 bp

.TATA box

R) UASc -210 -193 ATGCGTCTTTTCCGCTG...

HI (coding!

i C:u.ZnSOD (non-coding)

ill I /II II/II

. ..AAGCGGCATTTGCGCTG... -124 -240

FIGURE 13. Comparison by computer-aided sequence analysis of the untranslated 5’ regions of the CuZn SOD and MT genes showing sequence similarities between CuZn SOD promoter region and MT UAS regions (ACE1 binding site). Numbering is with respect to the ATG start codon.

mutant strain lacking ACE1 grown under normal aerobic

and the corresponding conditions and under

wild type strain (DTY22) were the same conditions with addition

of Cu. In addition, the wild type strain D273-10B (see above) was grown under the same conditions, however, with silver replacing the added Cu. Determinations of the CuZn SOD-mRNA, immunoreactive CuZn SOD, and CuZn SOD activity levels suggested that CuZn SOD expression is indeed co-regulated with Cu MT. Further support for this interpretation of the results came from the facts 1) that it was possible to purify a silver-SOD from yeast grown in the silver-supplemented medium and 2) that computer analysis of the CuZn SOD promoter region showed a striking homology of the nucleotide sequence and positioning with respect to the Cu MT promoter in the regions interacting with ACE1 [ 1821 (Fig. 1.3). Joan S. Valentine then reported on experiments in which four of the his metal ligands of yeast CuZn SOD (his 120. 48, 46, and 80) were replaced by cys residues by site directed mutagenesis (Fig. 14). The all-cupric derivatives of each of these mutants were prepared and the UV and visible spectra were measured. Two different types of spectra were observed: the cys 120 and cys 46 Cu-site mutants are yellow and have new intense absorption bands (relative to wild type) at 400 and 179 nm.

/

“,SG,

FIGURE 14. Schematic drawing of the active site of CuZn SOD according to the structure of bovine SOD, but with residue numbering adapted for yeast SOD.


SYSTEMS:

MANZIANA

REPORT

211

respectively. The spectra resemble Cu thiolate model complexes with square, octahedral, or five-coordinate environments. The cys 48 Cu-site mutant and the cys 80 Zn-site mutant are green with bands at 370 and 600 nm (cys 48) and 406 and 602 nm (cys 80). These spectra resemble those of a few model complexes that are assumed to be square planar with equatorial thiolates, but are not structurally characterized. The cys 80 mutant contains a cys in the Zn binding site which is distorted tetrahedral in the wild type enzyme. Valentine concludes that the cys 80 mutant has some degree of flexibility, since it does not give the typical tetrahedral type-l Cu spectrum. When Co2+ is introduced into the Zn-site of the cys 80 mutant protein, the UV-visible spectrum is remarkably similar to those of Co-substituted type-l proteins, suggesting that the Zn-site of the cys 80 mutant is tetrahedral when Co2+ is bound there. Valentine then turned to the biological roles of Cu,Zn SOD in yeast. As in general with all SODS, their biological role remains unclear. Mutant strains of bacteria that lack Mn SOD and Fe SOD, and mutant strains of yeast that lack CuZn SOD are highly sensitive to oxygen or paraquat toxicity, and transformation of such sod strains, either with their own SOD genes or SODS from other organisms, will restore normal wild type resistance, i.e., the human CuZn SOD gene restores bacterial sod mutants and the bacterial Mn SOD gene restores yeast sod mutants to normal oxygen and paraquat resistance [ 183- 1851. On the other hand, Fee’s discovery that E. coli Mn SOD and Fe SOD genes are controlled by the ferric uptake regulation (fur) locus, suggest the possibility that these proteins play a role in iron metabolism [ 1861. It has been reported that Cu,Zn SOD levels in yeast are increased in response to addition of Cu. Cu is also known to induce Cu MT in yeast. In this latter case the mechanism is known, i.e., Cu binds to the ACE1 protein which then binds in a Cu-dependent fashion to the upstream activating sequence (UAS) on the yeast MT gene and induces transcription of the gene (see preceding lecture). The possibility was, therefore, explored that Cu induction of Cu,Zn SOD occurs by a similar mechanism. The results of experiments aimed at clarifying this point show clearly that ACE binds in a Cu-dependent fashion to a UAS in the promoter region of the yeast Cu,Zn SOD gene. The UAS on the Cu,Zn SOD gene has been localized. It shows a high degree of homology to the UAS in the MT promoter region [187], yet the significance of this finding with respect to the function of SOD is not clear at this point. The results reported by Valentine on this last topic complement nicely and support the conclusions drawn by the preceding speaker. In the final presentation of the meeting D. S. Sigman told the audience in a lively lecture of his pioneering work on “chemical nucleases,” with the main example being l,lO-phenanthroline-Cu (PCu), as expected at a copper meeting. Chemical nucleases in general are redox-active coordination complexes that nick nucleic acids under physiological conditions by oxidative attack on the ribose or deoxyribose moiety. l,lO-PCu can serve as a reliable footprinting reagent and is useful for defining functionally important sequence dependent conformational variability of DNA and protein-induced structural changes in DNA. The intrinsic specificity of the reagent can be overridden by linking it to deoxyoligonucleotides or DNA-binding proteins. One then observes site-specific scission, which reflects the binding site of the targeting ligand. Sigman convinced the audience that enzymes do not have an absolute lease on specificity, but that with ingenuity, out-of-the-bottle reagents can also be made remarkably specific. Since the work on chemical nucleases has just had prominent publicity in the readily accessible literature, the interested reader is referred to these sources [ 188- 1901.

212 I-L Beinert

As in the old tradition, the meeting ended with some overall commentary by the writer, reflections on past and expected future developments, brief summaries of the highlights of the meeting (as perceived by the writer), and thanks to all of those having provided funds, time, and effort to the realization of this event.

The meeting was generously supported by: The National Research Councii of Italy; The United States National Science Foundation, contract number INT. 9002878; the Univer.rities of Rome “La Sapienza” and ‘“Tar Vergata”; the Italian Society of Biochemistry; the Manziana Municipality; and the Mayor of Manziana, Dr. Piciurro. The meeting was organized by Bruno Mondovi and Jack Peisach and the smooth operation of the proceedings was made possible by the untiring tflorts of Pieritrig:l Kiccin, Luciu Marcocci, Paola Loreti, and Paola Pietrangeii; the paperwork M’IIIS experrcv handled by Enza Buganza and Laura De Marco.

REFERENCES 1. H. Beinert, Life Chem. Reports 5, 319-341 (1987). 2. H. Beinert, Coord. Chem. Revs. 33, 55-85 (1980). 3. B. Mondovi, in Structure and Functions of Amine Oxidases. R. Mondovi. Ed., CRC Press, Boca Raton. Fla., 1985, preface, Table 1. 4. S. M. Janes, D. Mu, 1~).Wemmer, A. J. Smith, S. Kaur. D. Maltby, A. L. Burlingame, and J. P. Klinman, Science 248, 981-987 ( 1990). B. Mondovi and P. Rtccio, Biol. Metals 2, 110-l 13 (1990). L. Morpurgo, E. Agostinelli. B. Mondovi. L, Avigliano. R. Silvestri, G. Stefancich. and M. Artico, Biochemistry, submitted. J. P. Klinman, D. M. Dooley, J. A. Duine. P. F. Knowles. B. Mondovi. and J. J. Villafranca. FEBS Lett.. 282, i-4 (1991). C. I.. Lobenstein-Verheek. J. A. Jongejan, J. Frank, and J. A. Duine. FEBS Lett. 170.

305-309 (1984). 9. R. A. Van der Meer, J. A. Jongejan,

J. Frank, and J. A, Duine. FEBS Lett. 206. 111-114 (1986). 10. J. Van Iersel, R. A. Van der Meer, and J. A. Duine, Eur. J. Biochem. 161, 415-419

(1986~. 11. R. A. Van der Meer and J. A. Duine, Biochem. .I. 239, 789-791 (1986). 12. A. Y. Chistoserdov, Y. D. Tsygankov, and M. E. Lidstrom. Biochem. Biophys. Res. Comm. 172, 2 11-216 (1990). 13. M. M. Whittaker and J. W. Whittaker, J. Bioi. Chem. 265, 9610-9613 (1990). 14. J. G. Robertson, A. Kumar, J. A. Mancewicz. and J J. Villafranca. .I. Biol. C’hem. 264, 19916- 19921 (1989). 15. W. S. McIntire. J. 1.. Bates, D. E. Brown, and D, M, Dooley, Biochemistry 30,

125-133 (1991). H. Seno. T. Urakami, and 0. Suzuki, Arch. Biochem. Bioph.ys. 283. 533-536 (1990). 17. J. A. Duine, Trends Biochem. Sci. 16, 12 (1991). 18. N. Ito, S. E. V. Phillips, C. Stevens, Z. B. Ogel, M. J. McPherson, J. N. Keen, K. D. S. Yadav, and I’. F. Knowles, Nature 350, 87-90 (1991).

16. T. Kumazawa,

19. M. A. Paz, R. Fluckiger. A. Boak, H. M. Kagan, and P. M. Gallop, J, Rio/. Chem. 266, 689-692 (1991). 20. G. Banchelli, F. Buffoni. J. Elliott, and B. A. Callingham, .Neurochem. Int. 17,

215--221 (1990).


SYSTEMS:

MANZIANA

REPORT

213

21. F. Buffoni and S. Cambi, Anal. Biochem. 187, U-50 (1990). 22. F. Buffoni, Biochim. Biophys. Acta 1040, 77-83 (1990). 23. C. Hartmann and J. P. Klinman, J. Biof. Chem. 262, 962-965 (1987). 24. C. Hartmann and J. P. Klinman, FEBS Lett. 261, 441-444 (1990). 25. D. M. Dooley, M. A. McGuirl, D. E. Brown, P. N. Turowski, W. S. McIntire, and P. F. Knowles, Nature 349, 262-264 (1991). 26. D. Collison, P. F. Knowles, F. E. Mabbs, F. X. Rius, I. Singh, D. M. Dooley, C. E. Cote, and M. McGuirl, Biochem. J. 264, 663-669 (1989). 27. D. M. Dooley, M. A. McGuirl, C. E. Cote, P. F. Knowles, I. Singh, M. Spiller, R. D. Brown III, and S. H. Koenig, J. Am. Chem. Sot. 113, 754-761 (1991). 28. S. Suzuki, T. Sakurai, A. Nakahara, T. Manabe, and T. Okuyama, Biochemistry 25,

338-341 (1986). 29. L. Morpurgo, E. Agostinelli, 0. Befani, and B. Mondovi, Biochem. J. 248, 865-870 (1987). 30. M. M. Whittaker and J. W. Whittaker, J. Biol. Chem. 263, 60746080 (1988). 31. L. Morpurgo, E. Agostinelli, J. Muccigrosso, F. Martini, B. Mondovi, and L. Avigliano, Biochem. J. 260, 19-25 (1989). 32. L. Morpurgo, E. Agostinelli, B. Mondovi, and L. Avigliano, Biol. Metals 3, 114-117 (1990). 33. M. Artico, F. Corelli, S. Massa, G. Stefancich, L. Avigliano, 0. Befani, G. Marcozzi, S. Sabatini, and B. Mondovi, J. Med. Chem. 31, 802-806 (1988). 34. I. Savini, S. D’Alessio, A. Giartosio, L. Morpurgo, and L. Avigliano, Eur. J.

Biochem. 190, 491-495 (1990). 35. M. C. Bonaccorsi di Patti, G. Musci, A. Giartosio, S. D’Alessio, and L. Calabrese, J. Biol. Chem. 265, 21016-21022 (1990). 36. A. Giartosio, E. Agostinelli, and B. Mondovi, Biochem. Biophys. Res. Comm. 154, 66-72 (1988). 37. M. M. Whittaker, V. L. DeVito, S. A. Asher, and J. W. Whittaker, J. Biol. Chem. 264, 7104-7106 (1989). 38. K. Clark, J. E. Penner-Hahn, M. M. Whittaker, and J. W. Whittaker, Am. Chem. sot. 112, 6433-6434 (1990). 39. S. 0. Pember, J. J. Villafranca, and S. J. Benkovic, Biochemistry 25, 6611-6619

(1986). 40. S. 0. Pember, S. J. Benkovic, J. J. Villafranca, M. Pasenkiewicz-Gierula, and W. E. Antholine, Biochemistry 26, 4477-4483 (1987). 41. J. McCracken, S. Pember, S. J. Benkovic, J. J. Villafranca, R. J. Miller, and J. Peisach, J. Am. Chem. Sot. 110, 1069-1074 (1988). 42. S. 0. Pember, K. A. Johnson, J. J. Villafranca, and S. J. Benkovic, Biochemistry 28, 2123-2130 (1989). 43. A. Onishi and S. J. Benkovic, J. Biol. Chem., in press (1991). 44. R. A. Scott,

R. J. Sullivan,

W. E. De Wolfe,

R. E. Dolle,

and L. I. Kruse,

Biochemistry 27, 5411-5417 (1988). 45. N. J. Blackbum, T. M. Pettingill, K. S. Seagraves,

and R. T. Shigeta, J. Biol. Chem. 265, 15383-15386 (1990). 46. T. M. Pettingill, R. W. Strange, and N. J. Blackbum, J. Biol. Chem., in press (1991). 47. J. McCracken, P. R. Desai, N. J. Papadopoulos, J. J. Villafranca, and J. Peisach,

Biochemistry 27, 48. W. E. Blumberg, Biol. Chem. 264, 49. G. K. Farrington,

4133-4137 (1988). P. R. Desai, L. Powers,

J. H. Freedman,

and J. J. Villafranca,

J.

6029-6032 (1989). A. Kumar, and J. J. Villafranca,

J. Biol. Chem. 265, 1036-1040

(1990). 50. J. G. Robertson, P. R. Desai, A. Kumar, G. K. Farrington, Villafranca, J. Biol. Chem. 265, 1029-1035 (1990).

P. F. Fitzpatrick,

and J. J.

214

H. Beinert

51. A. H. Bertelsen, G. A. Beaudry, E. A. Galella, B. N. Jones, M. L. Ray, and N. M. Mehta, Arch. Biochem. Biophys. 279, 87-96 (1990). 52. G. A. Beaudry, N. M. Mehta, M. L. Ray. and A. H. Bertelsen. J. Viol. Chem. 265, 17694417699 (1990). 53. P. P. Tamburini, S D. Young, B. N. Jones, R. A. Palmesino. and A P. Consalvo. Int. J. Peptide Prolein Res. 35, 153-156 (1990). 54. B. A. Eipper, R. E. Mains, and E. Herbert, Trends Neurosci. 9. 463.-368 (198ht. 55. D. J. Merkler and S. D. Young, Biochemistrv. in press (1991). 56. C. Southan and I.. I. Kruse, FEBS Letl. 255. 116-120 (1989). 57. S. G. Sharoyan and R. M. Nalbandyan, Neurochimiu 7. 389-396 (1988). 58. K. A. Markossian. N A. Paitian. and R. M. Nalbandyan. Hiochimia 54. 2046-2053 (1990). 59. S. G. Sharoyan and R. M. Nalbandyan, FEBS Lett.. in press. 60. S. Turley, E. T. Adman, I_. C. Sieker, M.-Y. Liu. W J. Payne, and J. LeGall, J

Mol. Biol. 200, 417-419 (1988). 61. J. Godden, S. Turley. E, T. Adman. M.-Y. Liu, W. I. Payne, and J. LeGall. Scirnce. 253 (1991). 62. A. Messerschrnidt and R. Huber, Eur. J. Biochem. 187, 331-352 (1990). 63. F. Fenderson, S. Kumar, E. T. Adman, M.-Y. Liu. W, J. Payne. :tnd J. LcCall.

Biochemistry, 30, 7180-7185 (1991). 64. J. Riester. W. G. Zumft, and P. M. H. Kroneck. (1989). 65. P. M, H. Kroneck,

J, Riester,

Eur

J. Biochern. 178. 751 -762

W. G. Zumft, and W E Antholinr.

Bioi. Meta/.s 3.

103- 109 (1990). 66. P. M. H. Kroneck, 67. 68. 69. 70. 71. 72.

73. 74. 75.

76. 77.

78. 79.

80. 8 1.

82.

W. E. Antholine, D. H. W. Kastrau. G. Bust, G. (‘. hl. St&ens. and W. G. Zumft. FEBS Lett. 268, 274-276 (1990). G. L. Yewey and W. S. Caughey. ,4nn. ,ven York Acad. Sci. 550, 22-32 (1988). L. P. Pan, Z. Li, R. Larsen. and S. I. Chan, J. Rio/. C’hem. 266. 1367- I370 ( 1991). K. Lerch. M. Huber, Il. J. Schneider, R. Drexel, and B. Linzen. .I. Znnrg. Biochem. 26. 213 -217 (1986). M. Beltramini, B. Salvato. M, Santamaria, and K. 1,erch. Riochirn. Hioph.vs. .4ctci 1040, 365-372 (1940) W. Voll and R. Voit, Proc. Natl. Acad. Sci. USA 87, 5312-5316 (1990). E. Willott, X. Y. Wang, and M. A. Wells, J. Biof. (‘hem. 264. 19052 19059 (1989). T. Fujii. H. Sakurai. S. Izumi, and S. Tomino. J. Hid Chem 264. 11020~ 11025 (1989). B. Salvato and M. Beltramini, Life Chem. Rep. 8. I -47 (1990). J.-P. Tahon, G. Maes, C. Vinckier, R. Witters. ‘I‘. Zeegers-Huyskens. M. De Lev. and R. Lontie, Biochern. J. 271, 779-783 (1990). B. Salvato. M. Beltramini. A. Piazzesi. M. Alviggi. F. Ricchelli. K. S. Magliozzo. and J. Peisach, Inorg. Chim. Acta 125, 55-62 (1986). J. Lorosch and W. Hadse, Biochemistry 25. 5850-5857 (1986). T. J. Dutton, T. F Baumann. and J A. Larrahee. Inore. Chn. 29. 2272 -227X (1990). C. Ruegg and K. Lerch, Biochemistry 20. 1256.-1262 (I98i1. A. Bianconi and A. C’ Castellano. Eds., Biophys nr7d Synchratron Hadiat. 2. (1987). A. Bianconi, XANES Spectroscopy it7 X-ra.v A hsorption: Principles 1 Appkations. Techniques of EXAFS. SEXAFS. and XANES, I). C. Koningsbrrger and R. Prim, Eds., J. Wiley & Sons. New York, 1988, p. 573. W. B. Mims and J. Peisach. in Advanced EPR in Bioiogv und Biochemistry. A. Hoff. Ed.. Elsevier. Amsterdam. 1989. pp. i 57.


SYSTEMS:

MANZIANA

REPORT

215

83. W. B. Mims and J. Peisach,

J. Biol. Chem. 254, 4321-4323 (1979). 84. W. B. Mims and J. Peisach, J. Chem. Phys. 19, 4921-4930 (1978). 85. F. Jiang, J. McCracken, and J. Peisach, J. Am. Chem. Sot. 112, 9035-9044 (1990). 86. M. Colaneri, J. A. Potenza, H. J. Schugar, and J. Peisach, J. Am. Chem. Sot. 112, 9451-9458 (1990). 87. H. Thomann and M. Bemardo, Adv. in Chem., Am. Chem. Sot., Wash., 229 (1991). 88. H. Thomann and M. Bemardo,

Spectrosc. Znt. J. 8, 117 (1990). 89. H. Thomann and M. Bemardo, Chem. Phys. Lett. 169, 5 (1990). 90. H. Thomann, M. Bernardo, M. Lowry, M. Baldwin, and E. I. Solomon, J. Am. Chem. Sot. 113, 5911-5913 (1991). 91. M. D. Allendorf, D. J. Spira, and E. I. Solomon, Proc. Natl. Acad. Sci. USA 82, 3063-3067 (1985). 92. L. Morpurgo, A. Desideri, and G. Rotilio, Biochem. J. 207, 625-627 (1982). 93. L. Morpurgo, A. Desideri, and G. Rotilio, in The Coordination Chemistry of Metalloenzymes, I. Bertini, R. S. Drago, and C. Luchinat, Eds., Reidel Publishing Co., Dordrecht, Holland, 1983, pp. 207-213. 94. C. D.LuBien, M. E. Winkler, T. J. Thamann, R. A. Scott, M. S. Co, K. 0. Hodgson, and E. I. Solomon, J. Am. Chem. Sot. 103, 7014-7016 (1981). 95. A. Messerschmidt, A. Rossi, R. Ladenstein, R. Huber, M. Bolognesi, G. Gatti, A. Marchesini, R. Petruzzelli, and A. Finazzi-Agro, J. Mol. Biol. 206, 513-529. 96. D. J. Spira-Solomon, D. M. Allendorf, and E. I. Solomon, J. Am. Chem. Sot. 108, 5318-5328 (1986). 97. L.-S. Kau, D. J. Spira-Solomon, J. E. Penner-Hahn, K. 0. Hodgson, and E. I. Solomon, J. Am. Chem. Sot. 109, 6433-6442 (1987). 98. D. J. Spira-Solomon and E. I. Solomon, J. Am. Chem. Sot. 109, 64216432 (1987). 99. J. L. Cole, C. 0. Tan, E. K. Yang, K. 0. Hodgson, and E. I. Solomon, J. Am. Chem. Sot. 112, 2443-2249 (1990). 100. T. D. Westmoreland, D. E. Wilcox, M. J. Mims, W. B. Baldwin, and E. I. Solomon, J. Am. Chem. Sot. 111, 6106-6123 (1989). 101. A. A. Gewirth and E. I. Solomon, J. Am. Chem. Sot. 110, 3811-3819 (1988). 102. J. L. Cole, P. A. Clark, and E. I. Solomon, J. Am. Chem. Sot. 112, 9534-9548 (1990). 103. N. Kitajima, K. Fujisawa, and Y. Moro-Oka, J. Am. Chem. Sot. 111, 8975-8976 (1989). 104. N. Kitajima, T. Koda, Y. Iwata, and Y. Moro-Oka, J. Am. Chem. Sot. 112, 8833-8839 (1990). 105. P. K. Ross and E. I. Solomon, J. Am. Chem. Sot. 112, 5871-5872 (1990). 106. P. K. Ross and E. I. Solomon, J. Am. Chem. Sot., 113, 3246-3259 (1991). 107. Z. Tyeklar and K. D. Karlin, Accts. Chem. Res. 22, 241 (1989). 108. Z. Tyeklar, P. P. Paul, R. R. Jacobson, A. Farooq, K. D. Karlin, and J. Zubieta, J. Am. Chem. Sot. 111, 388 (1989). 109. K. D. Karlin and Y. Gultneh, Prog. Znorg. Chem. 35, 219 (1987). 110. L. Casella, M. Gullotti, E. Suardi, M. Sisti, R. Pagliarin, and P. Zanello, J. Chem.

Sot. Dalton Trans., 2843 (1990). 111. L. Casella, M. Gullotti, A. Pintar, F. Pinciroli, R. Vigano, and P. Zanello, J. Chem. Sot. Dalton Trans., 1161 (1989). 112. L. Casella, M. Gullotti, G. Pallanza, and L. Rigoni, J. Am. Chem. Sot. 110, 4221-4227 (1988). 1668-1669 (1985). 113. L. Casella and L. Rigoni, J. Chem. Sot., Chem. Commun., 114. 0. J. Gelling, F. van Bolhuis, A. Meetsma, and B. L. Feringa, J. Chem. Sot., Chem.

Commun.,

552 (1988).

216

H. Beinert

115. L. Casella, M. Gullotti, and G. Pallanza, 1 16. M. W. Mather, P. Springer, and J. A. G. Hauska and R. Thauer, Eds. Springer, 117. B. H. Zimmermann, C. I. Nitsche, J. A.

Biochem. Sot. Trans. 16. 821-822 (lY88). Fee, Proc. 41st Geo. Biol. Chem. Coil.. Berlin, pp. 94 - 104. (1990). Fee. F. Rusnak. and E. Miinck. Proc iVat/.

Arad. Sci. USA 85, 5779-5783 (1988). 118. J. A. Fee, M. W. Mather,

P. Springer.

S. Hensel.

and G. Bust.

Ann.

.VPW

York

Acnd. Sci. 5.50. 3.7--38 (1988). 119. M. W. Mather, P. Springer, and J. A. Fee, 1. Biol. Chem. 266, 502% 503.5 ( 199 1). 120. F. Malatesta, G. Antonini, P. Sarti, B. Vallone. and M. Bnmori. Hiol. Meta/.r. 3. 118-121 (1990). 105. 121. M. Numata, T. Yamazaki, Y. Fukumori, and T. Yamanaka. ,I Biochem. 245-248 (1989). 122. K. Kobayashi. H. Une. and K. Hayashi. J. Biol. Chem. 264, 797% 7Y80 ( 1989 1. 123. J. E. Morgan, P. M. Li. D.-J. Jang, M. A. ElSayed, and S. Ghan. Biochemistrr 28. 6975-6983 (1989). 124. F. Malatesta, P. Sarti, G. Antonini, B. Vallone. and M. Brunori. Proc. R;atl. .-lead. Sci. USA 87, 7410-7413 (1990). 125. M. C. Silvestrini. M. G. Tordi, G. Musci, and M. Bnmori. .I. Biol. Chem 265. 11783- 11787 (1990). 126. E. T. Adman, in T0pic.s in Molecular and Structural Biology. ?Cfetailoproteins. Vol. 1. P. M. Harrison, Ed., New York, MacMillan, 1986, pp. 1.~42. 127. C. M. Groeneveld and G. W. Canters, J. Biol. Chem. 263, 167 173 (1988). 128. M. van de Kamp, M. C. Stlvestrini, M. Brunori, J. van Bceumen, F C. Hali. and G. W. Canters. Eur. J. Biochem., 194, 109-118 (19901 129. M. van de Kamp. R. Flori\, F C. Hali, and G. W. Canter-s. .I Am. (‘hem. Sot. 112. 907-908 (1990). 130. A. Lommen and G. W. Canters, J, Biol. Chem. 265. 2768-2774 (1990). 131. A. Lommen. K. I. Pandya. D. C. Koningsberger. and G W Canters. Biochim.

Biophys. Acta 1076. 439-447 (1990). 132. H. Nar, A. Messerschmtdt, R. Huber. M. van de Kamp. and G. W. Canters. .1. Zfoi. Biol.. 218, 427-447 i 1991). 133. A. J. Lommen, A. Ratsma, N. Bijlsma, G. W. Canters. 9. E. van Wielink. J Frank. and J. van Beeumen, Eur. J. Biochem. 192, 653-661 (1990). 134. T. Sakurai, 0. Ikeda, and S. Suzuki, lnorg. Chem. 29, 4715-4718 (1990). 135. 0. Farver and I. Pecht, Proc. Nat!. Acad. Sci. USA 86. 6968.-6972 (1989). 136. 0. Farver and I. Pecht, Mol. Cryst. Liquid Cryst., in press (1990). 137. J. N. Onuchich and D. N. Beratan, /. Chem. Phys. 92. 722-733 (1990). 138. L. Morpurgo. G. Rotilio. A, Finazzi-Agro. and B. Mondovi. Biochim. Biophys. Acra 336, 324 -328 (1974). 139. J. Morpurgo. I.. Calabrese, A. Desideri, and G. Rotiiio. Biochrm. .I. 193. 639.-642 (1981). 140. L. Morpurgo, E. Agostinelli, M. Senepa, and A. Desideri, J Inorg. Biochem. 24, l-8 (1985). 141. M. M. Morie-Bebel, M. C. Morris. J. L. Menzie. and D R. McMillin. J. Am Chem. Sot. 106, 3677-3678 (1984). 142. A. S. Klemens, D. R. McMillin, H. T. Tsang, and J. E. Penner-Hahn. .I. Amer. Chem. Sot. 111, 639886402 (1989). 143. J. C. Severns and D. R. McMillin, Biochemistry 29, 8592-8597 (1990). 144. M. T. Graziani, P. Loreti, L. Morpurgo, I. Savini, and L. Avighano, bnorg. Chim.

Acta 173, 261--264 (1990) 145. L. Morpurgo. I. Savini, B. Mondovi, 25.-31 (1987).

and L. Avigliano.

.I. Inorg.

Biochem.

29,


146. M. T. Graziani,

L. Morpurgo,

SYSTEMS:

G. Rotilio,

MANZIANA

and B. Mondovi,

REPORT

217

FEBS Lett. 70, 87-90

(1976). 147. L. Morpurgo,

H.-J. Hartman, A. Desideri, U. Weser, and G. Rotilio, Biochem. J. 211, 515-517 (1983). 148. J. E. Penner-Hahn, B. Hedman, K. 0. Hodgson, D. J. Spira, and E. I. Solomon,

Biochem. Biophys. Res. Commun., 119, 567-574 (1984). 149. M. A. Mateescu, G. Weltrowska, E. Agostinelli, R. Saint-Andre, M. Weltrowski, and B. Mondovi, Biotechnol. Tech. 3, 415-420 (1989). 150. J. Brandt, L. 0. Andersson, and J. Porath, Biochim. Biophys. Acta 386, 196-202 (1975). 151. M. A. Mateescu, E. Agostinelli, G. Weltrowska, M. Weltrowski, and B. Mondovi, Biology of Metals 3, 98-102 (1990). Biochim. Biophys. Acta 445, 579-597 152. L.-E. Andreasson and B. Reinhammar, (1976). 153. L.-E. Andreasson, R. Brand&, and B. Reinhammar, Biochim. Biophys. Acta 438, 370-379 (1976). 154. M. Esaka, T. Hattori, K. Fujisawa, S. Sakajo, and T. Asahi, Eur. J. Biochem. 191, 537-541 (1990). 155. G. Chichiricco, M. P. Cent, A. D’Alessandro, A. Oratore, and L. Avigliano, Plant Science 64, 61-66 (1989). 156. G. D’Andrea, M. Maccarrone, A. Oratore, L. Avigliano, and A. Messerschmidt, Biochem. J. 264, 601-604 (1989). 157. L. Calabrese, M. Carbonaro, and G. Musci, J. Biol. Chem. 263, 6480-6483 (1988). 158. G. Musci, M. Carbonaro, A. Adriani, A. Lania, A. Galtieri, and L. Calabrese, Arch. Biochem. Biophys. 279, 8-13 (1990). 159. R. Wever, F. X. R. Leeuwen, and B. F. van Gelder, Biochim. Biophys. Acta 302, 236-239 (1973). 160. P. Furst, S. Hu, R. Hackett, and D. Hamer, CeN 55, 705-717 (1988). 161. P. Furst and D. Hamer, Proc. Nat/. Acad. Sci. USA 86, 5267-5271 (1988). 162. V. C. Culotta, T. Hsu, S. Hu, P. Furst, and D. Hamer, Proc. Natl. Acad. Sci. USA 86, 3877-3881 (1989). 163. R. Kambadur, V. C. Culotta, and D. Hamer, Proc. Natl. Acad. Sci. USA 87, 9168-9172 (1990). 164. I. Bertini, C. Luchinat, L. Messori, and M. Vas& J. Amer. Chem. Sot. 111, 7296-7300 (1989). 165. I. Bertini, C. Luchinat, L. Messori, and M. Vasfik, J. Amer. Chem. Sot. 111, 7300-7303 (1989). 166. I. Bertini, C. Luchinat, L. Messori, and M. Vas& J. Znorg. Chem., in press (1991). 167. K. Djinovic, G. Gatti, A. Coda, L. Antolini, G. Pelosi, A. Desideri, M. Falconi, F. Marmocchi, R. Rotilio, and M. Bolognesi, Acta Crystallographica, in press (1991). 168. A. Desideri, M. Falconi, V. Parisi, and G. Rotilio, FEBS Lett. 250, 45-48 (1989). 169. M. Falconi, G. Rotilio, and A. Desideri, Proteins, in press (1991). 170. J. J. Sines, S. A. Allison, and J. A. McCammon, Biochemistry 29, 9403-9412 (1990). 171. F. Paoletti and A. Mocali, Cancer Res. 48, 6674-6677 (1988). 172. S. S. Percival and E. D. Harris, Am. J. Physiol. 258, C140-Cl46 (1990). 173. M. Ciriolo, A. Desideri, M. Paci, and G. Rotilio, J. Biol. Chem. 265, 11030-11034 (1990). 174. C. Steinkuhler, 0. Sapora, M. T. Carri, W. Nagel, L. Marcocci, U. Weser, and G. Rotilio, J. Biof. Chem., in press (1991). 175. K. Felix, W. Nagel, H.-J. Hartmann, and U. Weser, Biol. Metals 2, 50-54 (1989). 176. B. Krauter, W. Nagel, H.-J. Hartmann, and U. Weser, Biochim. Biophys. Acta

1013, 212-217 (1989).

218

H. Beinert

177. K. Felix, W. Nagel, H.-J. Hartmann, and U. Weser, Biol. Metals 3. 141-145 (1990). 178. K. Felix and U. Weser, Biochem. J. 252, 577-581 (1988). 179. H.-J. Hartmann, T. Schechinger, and LJ. Weser. Biol. Metals 2, 40--44. (1989’1. 180. R. Miesel, H.-J. Hartmann. and U. Weser, Inflammation 14. 471.-483 (1990). 181. F. Galiazzo. M. R. Ciriolo. M. T. Carri, P. Civitareale. I,. Marcocci, F. Marmocchi, and G. Rotilio, Eur. /. Biochem., 196. 545-549 (1991). 182. M. T. Carri. F. Galiazzo, M. R. Ciriolo, and G. Rotilio. FEBS Lett. 278. 263.-266

(1991). 183. D. 0. Natvig,

K. Imlay, D. Touati, and R. A. Hallewell. J. Rio/ Chem. 262. 14697- 14701 (19871. 184. 0. Bermingham-McDonogh, E. B. Gralla. and J. S. Valentine. Proi~. Xati Sci. Arad. 85. 4789-4793 (1988). 185. C. Bowler, L. Van Kaer, W. Van Camp. M. Van Montagu. D. Inze. and P. Dhaese. J.

Bacterial. 172, 1539- 1546 (1990). 186. E. C. Niederhoffer, C. M. Naranjo. K, I,. Bradley. and J. A Fee. .I. Bucteriol. 172, 1930- 1938 (1990). 187. D. J. Thiele. Mol. and Ceil. Biol. 8, 2745-2752 (1988). 188. Ch.-B. Chen and D. S. Sigman, Science 237, 1197-1201 (1987). 189. D. S. Sigman and Ch.-B. Chen, Ann. Rev. Biochem. 59, 207 -236 11990). 190. D. S. Sigman, Biochemistp 29. 9097 -9105 (1990).

Received April 12, 1991; accepted Ma.y 6, 1991

Conference Report: SIXTH BIENNIAL SYMPOSIUM ON OPTICAL FIBER MEASUREMENTS Boulder, CO September 11-12, 1990.

Conference report: workshop on the role of the placenta in HIV infection and therapy, Nantucket, Massachusetts, 10-13 September 1990.

Conference Report: SIXTH INTERNATIONAL CONFERENCE ON THE APPLICATION OF STANDARDS FOR OPEN SYSTEMS, Gaithersburg, MD, October 2-4, 1990.

September 14, 1990: the beginning.

Report from the fourth nordic Conference on Family-Focused Nursing, Odense, Denmark, September 24-25, 2014.

Conference report: International Conference on Placenta--Tokyo, 1-3 October 1990.

Conference report: IVth meeting of European Placenta Group (EPG): joint with the Rochester Trophoblast Conference (RTC), 22-26 September 1991.

6th Dysferlin Conference, 3-6 April 2013, Arlington, Virginia, USA.

Conference Report: INTERNATIONAL CONFERENCE ON THE CHEMISTRY OF ELECTRONIC CERAMIC MATERIALS Jackson, WY August 17-22, 1990.

Abstracts of poster and platform presentations: Fourth International Conference on Computerized and Quantitative EMG. Mainz, Germany, September 13-15, 1990.

The beginning of metallurgy in the southern Levant: a late 6th millennium CalBC copper awl from Tel Tsaf, Israel.

Report of the Consensus Conference on Acne Classification. Washington, D.C., March 24 and 25, 1990.

Conference Report: SYMPOSIUM ON OPTICAL FIBER MEASUREMENTS Boulder, CO September 15-17, 1992.

Conference report. 6th International Symposium on Mass Spectrometry in Biochemistry and Medicine. Venice (Italy) 21-22 June 1979.

Abstracts from the FEBS EMBO 2014 Conference, 30 August-4 September, 2014, Paris, France.

Demyelination--background and mechanisms. Kyoto, Japan, 9-11 September 1990.

Abstracts from the 2nd International Joined OTIS - ENTIS Conference, September 18-21, 2014, Toronto, Canada.

Asia-Pacific Hematology Consortium Report on approach to multiple myeloma. Survey results from the 6th International Hematologic Malignancies Conference: Bridging the Gap 2015, Beijing, China.

Foreword to the special issue on selected papers from the 6th International Conference on Bioinformatics and Computational Biology (BICoB 2014).

Conference Report: WORKSHOP ON NEW MEASUREMENT TECHNOLOGY FOR POLYMER PROCESSING Gaithersburg, MD December 3-4, 1990.

6th Congress of the GTH (Gesellschaft für Thrombose- und Hämostaseforschung). Kiel, February 21-24, 1990. Abstracts.

Conference Report: SECOND INDUSTRY WORKSHOP ON POLYMER COMPOSITE PROCESSING, Gaithersburg, MD May 18, 1990.

Report from the 1st MYCOKEY International Conference Global Mycotoxin Reduction in the Food and Feed Chain Held in Ghent, Belgium, 11-14 September 2017.

Erratum to: 12th international congress of inborn errors of metabolism, 3rd-6th September, 2013.