ARCHIVES

Vol.

287,

OF BIOCHEMISTRY

No.

1, May

INVITED

15, pp.

AND

BIOPHYSICS

1-7,

1991

PAPER

Intra- and Intermolecular in Redox Proteins’ Gordon Department

Received

Electron Transfer

Tollin and James T. Hazzard of Biochemistry, University of Arizona,

December

3, 1990,

and

in revised

form

January

Tucson,

Arizona

APPROACH

We have developed a protocol for measuring intramolecular or intracomplex electron transfer rate constants in redox proteins which uses laser flash photolysis to rapidly (4 /.Ls)generate reducing equivalents in situ (in the form of flavin semiquinones) [cf. (1) and references cited therein]. Under the appropriate conditions, electron ’ The work described herein from the National Institutes ’ To whom correspondence

was supported in part of Health. should be addressed.

0003~9861/91 $3.00 Copyright ‘\L 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

85721

16, 1991

As is well known, electron transfer reactions play key roles in a wide variety of metabolic systems, including fundamental energy storage processes such as photosynthesis and respiration, as well as such widely diverse anabolic and catabolic transformations as nitrogen fixation, drug detoxification, sulfur assimilation, deoxyribonucleotide formation, and fatty acid synthesis and degradation. The enzymes which are involved have been studied intensively over many years, and a great deal is known about their structural and functional characteristics. It is not the purpose of the present review to provide a summary of this body of knowledge; indeed, to attempt to do this within the limitations of a journal article would be futile. Rather, we will focus on a single aspect of the biochemistry of these materials, which has been under investigation for the past several years in our laboratory, i.e., the structural features of redox enzymes which control the rate constants for electron transfer events occurring within 1:l complexes between two proteins, and within multisubunit proteins which possess more than one prosthetic group. The emphasis will be on questions which are still outstanding and future research directions, rather than on present achievements. EXPERIMENTAL

Processes

by Grant

DK15057

transfer from the flavin semiquinone will occur predominantly to one of the redox components of either a mixture of redox proteins or a multicenter redox protein, and it will then be possible to monitor subsequent electron transfer events in real time using spectrophotometry. The following set of equations describes the reaction sequence for a protein:protein electron transfer process: ha, EDTA

-

kL

FlH

FlH’ + (PI),, 2 Fl,, + (Pl)red + H+

[II Pal

FlH’ + (P&

Pbl

FL

2 Fl,,, + (PZ)red+ H+

(Pl)red+ WP)ox2

(PAX + (PAred,

[31

where Fl,, and FlH’ correspond to the oxidized and semiquinone flavin species, respectively, and Pi and P, refer to the lower and higher redox potential proteins, respectively. Equation [l] reflects the photochemical generation of the flavin semiquinone via triplet state quenching by the sacrificial electron donor, which is usually EDTA. Equations [2a] and [Zb] are depicted as parallel secondorder one-electron transfer reactions. The relative concentrations of (Pl)red and (PZ)redproduced are dependent upon the ratio of the respective rate constants and the relative concentrations of (PI),, and (PZ),, . Obviously, Reaction [3] will only be observed if reduction of the lower redox potential partner is favored. Thus, if kaa$ kzb and/

or [U’AJ

ti [U+‘A,,l, then [(PIM

% [(P2hl

and the

contribution of Eq. [2b] to the overall reaction sequence will be minimal. While this may seemcontradictory based on the general principles given below for electron transfer processes, we have observed in several systems that the higher redox potential component is not easily reduced by free flavin semiquinones, but undergoes facile reduction by its physiological protein partner. If this is not the case, 1

2

TOLLIN

AND

it is possible to enhance reduction of P, by making [(P&l % [ (P2)J. Under typical experimental conditions the concentration of FlH’ generated by the laser flash via Reaction [l] is relatively small ( 10 PM, pseudo-first-order conditions exist for Reactions [2a] and [2b]. The transfer of an electron from P, to PZ is depicted, in its most simple form, as a one-step bimolecular process in Eq. [3]. Plots of kobs vs [(PJ,,.J should be linear if Reaction [3] is a true second-order process (given that the reactions in Eqs. [l] and [2a] are sufficiently rapid so that they do not become rate limiting, which is a marked benefit of using photochemically generated flavin semiquinones as reductants). More often, however, hyperbolic plots are obtained for the concentration dependence of kobs. This suggests that the reaction mechanism of Eq. [3] must consist of a minimum of two steps, the initial formation of a collisional complex followed by a first-order process which controls the electron transfer reaction, and which becomes rate-limiting at high protein concentrations. This mechanism is shown in Eq. [3a] and [3b]. ha (P&d

+

[(Pl)red:(P2)oxl

(pP)ox

,c

3a[(Pl)red:(PZ)oxl

’

[(Pl)ox:(P2)redl

Pal [3bl

For reactions involving intramolecular electron transfer within electrostatically stabilized (i.e., those formed at very low ionic strength) or covalently linked 1:l protein complexes, or within a protein species containing more than one redox center, the electron transfer reaction should be simple first-order, i.e., concentration independent, as shown in Eq. [3b]. The cyt c-CcP3 system provides a good example of this approach (2,24). It is possible to observe the rapid reduction of cyt c by 5-deazariboflavin semiquinone at 550 nm, followed by the subsequent reduction of Compound I of CcP (i.e., the hydrogen peroxide oxidized species) at 557 nm (a cyt c isosbestic point), the latter occurring via electron transfer from reduced cyt c within a transient complex between the two proteins. Figure 1 shows examples of laser-induced transients obtained from this system. For reasons that are not completely understood at present, but which probably relate partially to the steric properties of the protein (see below), the direct reduction of CcP Compound I by deazariboflavin semiquinone occurs considerably more slowly (- lOOfold) than does reduction of oxidized cyt c. Thus, Eq. [2a] predominates over [2b] despite the fact that the midpoint ” Abbreviations used: AO, zucchini ascorbate oxidase; cyt c, cytochrome c; CcO, cytochrome c oxidase; CcP, cytochrome c peroxidase; FNR, ferredoxin-NADP+ reductase; LD, yeast lactate dehydrogenase (flavocytochrome b,); PCMH, p-cresol methylhydroxylase; PR, cytochrome P450 reductase; SO, sulfite oxidase; TMAD, trimethylamine dehydrogenase; XDH, xanthine dehydrogenase; X0, xanthine oxidase.

HAZZARD

potential for Compound I is 750 mV greater than that for cyt c. By carrying out these experiments as a function of CcP concentration, it is possible to determine a value for the limiting first-order rate constant for electron transfer within the 1:l complex between the two protein reactants. Chicken liver xanthine dehydrogenase provides an example of a multicenter redox protein for which intramolecular electron transfer rate constants can be determined by this procedure (3). In this case, deazariboflavin semiquinone transfers an electron exclusively to the molybdenum cofactor of XDH, which has the lowest redox potential of the various centers within this enzyme. By using the appropriate wavelengths it is then possible to follow subsequent electron transfer events from molybdenum to FAD and from FAD to one of the Fe&S& centers. Figure 2 shows the corresponding transients. Again, the dependence of the kinetics on protein concentration can be used to distinguish between bimolecular and intramolecular processes. GENERAL

CONSIDERATIONS

According to the theory of electron transfer developed by Marcus, and extended by others, the rate constant for outer sphere electron transfer between two redox centers (i.e., between two centers which do not share a common ligand) should principally depend upon the following factors (cf. (4) and references cited therein): i. Intercenter distance. An exponential distance dependence is predicted by theory (4). This has been reported in some model studies of protein electron transfer (5), although theoretical considerations (6), as well as some experimental results (7,8), have suggestedthe possibility of a more complex relationship between rate constant and distance in proteins, involving combinations of covalent bond, hydrogen bond, and through-space interactions. It is interesting to note that biological electron transfer reactions can occur over distances (215 A) greater than expected for direct outer-sphere electron transfer. ii. Intervening medium. In proteins, this may involve specific contributions from hydrogen bonds, aromatic side chains, thiol groups, etc. For electron transfer over large distances, the intervening medium probably plays a significant role in the overall process, although it has proven difficult to deduce probable pathways even within protein systems where the donor and acceptor locations are precisely known (8, 9). iii. Orientational effects. These will generally depend upon the orbital symmetries and geometries of the species involved in the electron transfer process. In someprotein: protein reactions direct outer sphere electron transfer may be possible (e.g., for proteins containing prosthetic groups having a high degree of solvent exposure), and thus a dependence on prosthetic group orientations might be significant. However, when distances are too great for outer sphere mechanisms, the significance of this param-

REDOX

PROTEIN

ELECTRON

TRANSFER

3

MECHANISMS

h=550nm h

5 ms

t

AA,

I

AA t

h =550nm 0.5

ms

I

d

I

b) h !

‘\ \ h=SOnm

AA f

5 ms

'

\

t

I

h=557nm 5 ms

decay curves obtained upon laser flash photolysis of solutions containing CcP Compound I and horse heart cytochrome c, in FIG. 1. Transient a buffer containing 100 mM phosphate (pH 6.0), 90 FM 5-deazariboflavin, and 0.5 mM EDTA. Data from Ref. (24). (a) Cytochrome c (30 pM) alone; monitoring wavelength 550 run. Increase in absorbance corresponds to reduction of the cytochrome c by deazariboflavin semiquinone. (b) CcP Compound I (20 pM) added to solution in (a); monitoring wavelength 550 run. Increase in absorbance again corresponds to cytochrome c reduction; this is followed by a monoexponential absorbance decrease due to CcP reduction and cytochrome c oxidation. (c) Same as in (b), except on a faster time scale to demonstrate kinetic separation between cytochrome c reduction and reoxidation. (d) Same as in (b), except monitoring wavelength was 557 nm, which is an isosbestic point for cytochrome c reduction. Absorbance decrease due solely to reduction of CcP Compound I. Observed rate constant obtained from these data is the same as that obtained from data in (b), thus demonstrating the occurrence of intracomplex electron transfer from reduced cytochrome c to CcP Compound I.

processes,and that it has not been possible as yet to study these one at a time. Unfortunately, no direct structure determinations have yet been made for an electron transfer complex between two redox proteins, although a complex between cyt c and CcP has been crystallized and its structure investigated by X-ray diffraction (10). In this case, however, the cyt c component was disordered in the crystal, suggesting the possible existence of multiple orientations. In the absence iv. Thermodynamic driving force, i.e., redox potential of direct measurement, computer modelling has been used differences. This has been clearly demonstrated in the in several cases(11-13) to generate hypothetical 1:l comreactions of small molecule reductants with small non- plexes, with salt link formation between oppositely enzymatic redox proteins, to a much lesser degree for re- charged side chains being the structural paradigm, based actions involving redox enzymes, and still less for protein: largely on the fact that in most cases complex formation protein reactions (cf. (1)). The latter is presumably due (albeit of an undefined nature) is known to be facilitated to the large contributions of other factors. Furthermore, by low ionic strength conditions. Other factors which have the “inverted” region, in which rate constants decrease been utilized in the model building exercises are related at sufficiently high driving force, which has been observed to the factors given above, i.e., prosthetic group separation with nonbiological reactions, has not as yet been shown distance and orientation, and the existence of residues to occur for protein:protein systems. which can serve as electron transfer conduits when the For reasons to be discussedbelow, it has proven difficult distances are too great for direct outer sphere electron to find relationships between biological electron transfer transfer. Whereas these models have proven to be useful reactions and the above-mentioned parameters. This for the design of kinetic experiments, the data obtained suggeststhat there are multiple factors controlling these from such experiments are sometimes in conflict with eter relates to the intervening medium (i.e., the protein matrix and/or solvent between the centers). Obviously, for interprotein reactions, distance, intervening medium, and orientational effects are interconnected, being dependent on the protein surface topographical features in the vicinity of redox cofactors, as well as on the distribution of electrostatic surface charges which will tend to have an orientational effect.

4

TOLLIN

(A)

! a

a

HAZZARD

1200,p

,

(B)

AND

r

0.24ms

aa

a

FIG. 2. Transient decay curves obtained upon laser flash photolysis of chicken liver xanthine dehydrogenase, in a buffer containing 100 mM phosphate (pH 7.8), 120 pM 5-deazariboflavin, and 20 mM EDTA. Data from Ref. (3). A, protein concentration, 17.5 pM; monitoring wavelength 483 nm, which corresponds to an isosbestic point for FAD reduction. Initial increase in absorbance in left hand trace corresponds to deazariboflavin semiquinone formation. Subsequent biphasic decrease corresponds to semiquinone reoxidation due to electron transfer to molybdenum center (fast phase) and reduction of iron sulfur center II (slow phase). Trace on right shows a still slower phase of iron sulfur center reduction. Solid curves correspond to single exponential fits to various portions of transient decays. All kinetic phases, except initial fast phase of absorbance decrease (shown in inset), are protein concentration independent, and thus intramolecular. B, protein concentration, 12.2 PM; monitoring wavelength 600 nm, which corresponds to FAD semiquinone absorbance. Solid curves correspond to single exponential fits. All kinetic phases are protein concentration independent. Absorbance increase in left hand trace results from reduction of FAD to the semiquinone form, due to electron transfer from molybdenum center. Absorbance decrease shown in right hand trace corresponds to FAD semiquinone reoxidation. Rate constant obtained from these data is the same as that obtained from fast phase of iron sulfur center reduction shown in (A); thus, these transients can be assigned to intramolecular electron transfer from FAD semiquinone to iron sulfur center II.

what might be expected based upon the hypothetical models (see below). Recently, Northrup et al. (14) have developed a complex formation algorithm which allows two proteins to diffuse toward each other, based on both Brownian motion and electrostatic interactions. The resulting simulations predict a family of collisional complexes, which suggestsa highly dynamic picture of transient electron transfer complex formation. ELECTROSTATIC EFFECTS ELECTRON TRANSFER

ON

INTRACOMPLEX

Direct measurements of the ionic strength dependence of intracomplex electron transfer rate constants for several physiological redox protein pairs (yeast or horse cyt c-CcP (15, 16), spinach ferredoxin-FNR (17), and bovine cyt c-Cc0 (18)) have demonstrated that electrostatic forces are not the sole determinant of optimal electron transfer rates. In all of these cases, the largest electron transfer rate constants were obtained at intermediate or

relatively high values of ionic strength, with rate constant values decreasing on either side of the optimum salt concentration. An interpretation of this type of dependence is that, at very low ionic strengths, the partners are “frozen” into a nonoptimal electrostatically stabilized orientation, and, at high ionic strengths, nonproductive complex formation becomes important. At the intermediate ionic strengths, other interactions such as hydrogen bonding and hydrophobic effects must be involved in mediating the formation of a complex which has optimal geometry for electron transfer. One should note, however, that it is clear from the ionic strength dependence that electrostatic forces are used to approximately orient the reactants in an appropriate manner. More work is required to determine precisely what these additional interactions are which are utilized in achieving the correct mutual orientation and distance following the initial collision event. Another indication of the importance of dynamic motions between redox partners in electron transfer com-

REDOX

PROTEIN

ELECTRON

plexes has been obtained from measurements of rate constants within complexes which have been covalently crosslinked between carboxyl and amino groups at low ionic strength. Presumably, cross-linking should lock the two proteins into the electrostatically stabilized, and hopefully most physiologically relevant, complex. However, in all of the systems examined thus far, which include cyt cCcP (16), flavodoxin-FNR (19), and cyt c-plastocyanin (20, 21), electron transfer rate constants were appreciably smaller for the covalent complex than for the transient complex formed upon collision at the optimum ionic strength. The most dramatic example is provided by the cyt c-plastocyanin system, in which intramolecular electron transfer is too slow to compete with intermolecular transfer between two molecules of the complex, whereas an intramolecular electron transfer rate constant of - 1000 s-’ was obtained for the noncovalent complex (20, 21). These results also provide the most direct illustration of the importance of mutual orientation, and perhaps also of flexibility within a 1:l complex, in determining electron transfer rates. This deserves further study, from both theoretical and experimental viewpoints. ROLE OF SPECIFIC AMINO INTRACOMPLEX ELECTRON

ACID SIDE TRANSFER

CHAINS

IN

We have used proteins containing single amino acid replacements, either naturally occurring revertants or genetically engineered mutants, in collaboration with the laboratories of J. Kraut (University of California, San Diego) and F. Sherman and G. McLendon (University of Rochester), to investigate the roles of specific amino acid side chains in controlling electron transfer rate constants within the yeast cyt c-CcP complex (22-24). The basis for choosing amino acids for modification was largely derived from the computer graphics model for the complex proposed by Poulos and co-workers (13), although one of the residues (Trp-191) was chosen on the strength of suggestions that it might be involved in the formation of the amino acid radical species which is generated in addition to the oxyferryl heme in Compound I by peroxide oxidation (25). Table I shows residues which were modified and the results of the kinetic measurements of intracomplex rate constants. The following conclusions can be drawn from these data. i. The results are partially consistent with the postulated interface contacts in the hypothetical complex, i.e., rate constants generally change appreciably when residues in these regions are modified. This can be stated with more certainty for cyt c than for CcP, inasmuch as in the latter case two residues postulated to have key roles in complex formation and/or electron transfer (Asp-217 and His-181) can be mutated with no or little effect on k,,. ii. Interestingly, native cyt c is not optimized for electron transfer to CcP, i.e., mutants are found which react more rapidly than the wild type. This is perhaps not sur-

TRANSFER

5

MECHANISMS TABLE

I

Amino Acid Substitutions on Intracomplex Electron Transfer Rate Constants between Yeast CcP CompoundI and Yeast Iso-1-Cytochrome c

Effects

of

kt (s-‘) Electrostaticmutants pH Cyt c species h-1 R181” Q2lK K77D

I I 7 7

I = 260

I=8mM

260 1000 100 440

mM

1460 2240 600 N.D.

k., K’) Structuralmutants CcP species* W.T. (UR)’ D37K D79K D217K

Structural

pH 6 6 6 6

mutants

pH

I =8

mM

134 793 3 111

I = 8

mM

I = 114

mM

1950

145 255 2053

I=30mM

I=

114mM

CcP speciesd Native Native W.T. (UCSD) W.T. (UCSD) W191F HlEilG

7 6 7 6 7 6

750 N.D. 850 N.D. no transfer N.D.

3220 N.D. 1350 N.D. detected N.D.

N.D. 4500 N.D. 3450 N.D. 1850

a For comparative purposes, yeast iso-l cyt c contains five additional residues at the N-terminus relative to tuna, horse, or bovine cyt c. Thus, these residues correspond to positions 13, 16, and 72 in the vertebrate cytochromes. The nomenclature for the mutants is to be read as follows: R181 is arginine at posit.ion 18 in the native protein mutated to isoleucine. Data from Ref. (22). * Reduction performed using yeast iso-l cyt c. Unpublished data obtained in collaboration with A. Corin, R. Hake, and G. McLendon of the University of Rochester and Kodak Corp. ‘The nomenclature for the CcP species is as follows. Native CcP corresponds to the enzyme isolated from baker’s yeast. The wild type (W.T.) enzymes are those species cloned into and expressed by E. coli; W.T. (UR) signifies the wild type cloned by Mr. Richard Hake in the laboratories of Drs. G. McLendon and A. Corin at the University of Rochester and Kodak Corp. D37K, D79K, and D217K are derived from this wild type. W.T. (UCSD) signifies the wild type cloned in the laboratory of Dr. J. Kraut at University of California, San Diego, by Drs. J. M. Mauro and M. A. Miller. H181G and W191F are derived from this parent strain. d Reduction performed using horse cyt c. Data from Refs. (23, 24).

prising, inasmuch as electron transfer is not the rate-determining step in the CcP mechanism, and cyt c must interact with Complexes III and IV of the respiratory pathway. Perhaps optimization has occurred with respect

6

TOLLIN

AND

to these latter systems. Further investigation will be necessary to establish this point. iii. Disrupting electrostatic interactions under low ionic strength conditions (I = 8 mM) by charge neutralization or reversal generally increases the rate constant for electron transfer, and vice versa, consistent with the ionic strength effects noted above (e.g., Arg-18, Gln-21, Lys32, Lys-77 mutations of cyt c and Asp-37 of CcP). Again, this points to the deleterious effect of too strong a mutual attraction between the reaction partners. iv. Conversion of three surface carboxylates on CcP to lysines has interesting effects. Whereas there is little or no effect of mutation of Asp-217 on electron transfer, charge reversal at Asp-37 reverses the ionic strength dependency of the rate constant relative to the wild type, and mutation of Asp-79, a residue far removed from any proposed interaction site, also has a profound effect on the magnitude of ket, although the ionic strength behavior is the same as for the wild type. In the case of Asp-79, this residue may play a fundamental role in electrostatic stabilization of the oxy-ferry1 heme in Compound I, which makes data interpretation more complex. Furthermore, in all three cases, we do not have X-ray crystallographic information, and thus structural perturbations affecting electron transfer remain a possibility. Further study is required. v. His-181 of CcP, postulated in the hypothetical complex (13) to play a key role in mediating electron transfer between the two proteins, is clearly not an obligatory component of the electron transfer pathway. However, Tip-191 of CcP, a residue which is deeply buried within the protein, clearly plays a crucial role in electron transfer. The nature of this role requires further study, however. Two possibilities are (a) it could be directly involved as a component of the electron transfer pathway and (b) its participation in a hydrogen-bonding network including the proximal histidine ligand to the heme iron may be a critical structural requirement for electron transfer. As is often the case when large effects are observed in mutagenesis experiments, multiple interpretations are possible, although it should be noted that in this case X-ray crystallographic results indicate the absence of large structural perturbations. The general perspective which emerges from these studies is that, despite the fact that this is arguably one of the most intensively characterized protein:protein electron transfer systems, we really understand very little about the structural features of CcP which allow both the formation of a productive electron transfer complex and a rapid rate for the electron transfer event. Much more work is clearly going to be required before we achieve a high level of insight into these features of the reaction mechanism. It should be noted, however, that in this case we can be fairly confident that specific structural characteristics of the proteins involved are crucial in allowing facile electron transfer. This conclusion derives from two

HAZZARD

observations: reduction of CcP Compound I by deazariboflavin semiquinone, a nonphysiological donor with a much lower redox potential than cyt c (-650 mV vs $250 mV) is quite sluggish (24), despite the large thermodynamic driving force (-+1.6 V); reduction of CcP Compound I by ruthenium atoms covalently attached to His residues which are located in regions of the protein far removed from the probable site of cyt c interaction, but which are at distances comparable to that of cyt c and which have larger driving forces, are also quite sluggish (26). Furthermore, complex formation of the ruthenated CCP with cyt c at low ionic strength does not alter the kinetics of intramolecular electron transfer, which indicates that cyt c binding does not induce a delocalized “conformational gating,” which could permit facile electron transfer from nonspecific regions of the CcP surface. An important question which arises from these observations is the following: does the interaction between cyt c and CcP result in structural changes which lead to the localized establishment of an effective electron transfer pathway, or does such a pathway already exist in free CcP, and is cyt c merely structured in such a way as to allow effective access to the appropriate region? Again, further study will be required to provide an answer to this question. INTRAMOLECULAR MULTICENTER

ELECTRON TRANSFER REDOX PROTEINS

IN

Relatively few X-ray structures are available for multicenter redox enzymes. If one excludes the bacterial photosynthetic reaction center, which can be thought of as a special case in which electron transfer proceeds at a high free energy level as a consequence of the absorption of light, those redox proteins for which structures have been determined, and for which values have been measured for intramolecular electron transfer rate constants, include the following: p-cresol methylhydroxylase (PCMH), trimethylamine dehydrogenase (TMAD), yeast lactate dehydrogenase (LD), and ascorbate oxidase (AO). Table II lists rate constant values for these proteins, along with a few others for which rate constants have also been obtained, as well as values for someof the critical parameters such as distance, orientation, and thermodynamic driving force (when these are known). It is clear from these data that no correlation can be established at the present time between electron transfer rate constants and any one factor, including thermodynamic driving force. Thus, multiple influences are certainly operative. It is also not possible at this time to determine whether electron transfer occurs via a through-space, a through-bond, or a mixed type of mechanism, although in some casesthe redox centers are close enough so that a through-space transfer involving direct orbital overlap is the most likely pathway (e.g., TMAD and PCMH). When the centers are further apart, as in LD or AO, this is more ambiguous. In the

REDOX

PROTEIN

ELECTRON

TRANSFER

7

MECHANISMS

TABLE11 Intramolecular Enzyme”

Reaction

TMAD x0 PCMH so PR LD A0

FMNH’ to 4.Fe-S FADH’to 2-Fe-S FADH’ to heme c MO to heme b FADH’ to FMN FMNH’ to heme b cu I to cu III

Electron

Transfer

Rate Constants

ket (s ~‘)

(mV)

AE”

62 97 220 310 70 400-1200’ 80

for Multicenter

Redox Enzymes

Distance (A)

Orientation

Ref.

5 9 8 ? 8-15 10 12

il b 66” b

(27) (28) (29) (30) (31) (32) (Xi)

60 40 =400 46 180 5 0

? 2o” h

’ TMAD: trimethylamine dehydrogenase; X0: milk xanthine oxidase; PCMH: Pseudomonas putida p-cresol methylhydroxylase; liver sulfite oxidase; PR: rabbit NADPH-cytochrome P450 reductase; LD: yeast lactate dehydrogenase; AO: zucchini ascorbate * It is not clear how to define orientation in these cases, inasmuch as at least one of the redox centers is not planar. ’ Rate constant ionic strength dependent.

case of LD, a possible candidate for mediating electron transfer is Tyr-143 (34). In AO, a more extensive connection between the type I and type III copper centers may exist, involving a copper-thiolate bond, a hydrogen bond from the carbonyl oxygen of Cys-A509 to Ns of His A508 and the imidazole ring of His-A508 (35). Future site-directed mutagenesis studies of these proteins may provide insight into such questions. In the meantime, however, we must conclude, as we did in the case of intracomplex electron transfer, that the present state of our understanding of the structural basis of intramolecular electron transfer in multicenter redox enzymes is relatively shallow. REFERENCES 1. Tollin,

Acta

15. Hazzard, ,J. T., McLendon, G., Cusanovich, M. A., and Tollin, G. (1988) Biochem. Biophys. Res. Commun. 151, 429.-434. 16. Hazzard, J. T., Moench, S. J., Erman, J. E., Satterlee, ,J. D., and Tollin, G. (1988) Biochemistry 27, 2002%2008. 17 Walker, M. C., Navarro, J. A., Pueyo, J. J., Gomez-Moreno, C., and Tollin, G. (1991) Arch. Biochem. Biophys. 287, 351-358. 18. Hazzard, ,J. T., Rong, 213-222. 19. Walker, M. C., Pueyo, Arch.

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and Kostic,

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24. Miller, M. A., Hazzard, J. T., Mauro, J. M., Edwards, S. L., Simons, P. C., Tollin, G., and Kraut, J. (1988) Biochemistry 27,9081-9088. 25. Edwards, S. L., Xuong, N. H., Hamlin, R. C., and Kraut, J. (1987) Biochemistry 26, 1503-1511. 26. Fox, T., Hazzard, J. T., Edwards, S. L., English, A. M., Poulos, T. L., and Tollin, G. (1990) J. Am. Chem. Sot. 112, 7426-7428.

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Sm.

D. N., and Onuchic, T. J., Gray,

J. N. (1989)

H. B., and Winkler,

Photosynth.

Res.

,J. R. (1989)

J. Am.

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