Volume 28, number 1-3

MOLECULARt~ CELLULAR BIOCHEMISTRY

D e c e m b e r 14, 1979

M O L E C U L A R ASPECTS OF CYTOCHROME c OXIDASE: STRUCTURE A N D DYNAMICS Angelo A Z Z I and R o b e r t P. C A S E Y Medizinisch-Chemisches Institut, Universit& Bern, Biihlstrasse 28, C H - 3 0 1 2 Bern~Switzerland (Received June 10, 1979)

Summary In the last few years much attention has been dedicated to the elucidation of some of the molecular aspects of cytochrome c oxidase. It has been shown conclusively that the enzyme from several sources (yeast, Neurospora, heart, liver) contains seven different subunits, which are asymmetrically inserted in the membrane. All of these are in contact with the lipid bilayer (except subunits V and VI) and to a greater or lesser extent with the water phase as well (except for subunit I). Subunit II of the enzyme appears to be involved in the formation of the binding site of cytochrome c. T h e location of the redox groups of the enzyme is still a matter of controversy. Their distance from the cytochrome c heme group is approximately 35 such that electron tunneling appears to be the only possible mechanism for transporting electrons across such a distance. A p r o t o n pump appears to be associated with electron transport and approximately one proton is extruded per electron equivalent reducing

Abbreviations: CCCP, carbonyl cyanide mchlorophenylhydrazone;DADH2, diamino-durene (reduced form); DCCD, N,N'-dicyclohexylcarbodiimide;FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone;Hepes, 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonicacid; NEM, N'-ethyl-maleimide; ApH, transmembrane chemicalpotential gradient of H+; At), transmembrane electricalpotential gradient; A/2I~, transmembrane electrochemicalpotential gradient of H+; Q/QH2, oxidised/reduced form of ubiquinone; TMPD/TMPDH2, non-protonated/protonated form of tetramethyl-p-phenylenediamine;SDS, sodium dodecyl sulphate.

oxygen via the enzyme. N,N', dicyclohexylcarbodiimide a well-established inhibitor of H ÷translocating ATPases inhibits the proton pump and labels specifically subunit III of the enzyme.

Introduction T h e structure and orientation of cytochrome c oxidase molecules in the inner m e m b r a n e of mitochondria has received special attention in recent years as a consequence of its fundamental role as terminal enzyme of the respiratory chain in most eukaryotes. The functional complexity of the enzyme, which transfers electrons; from the one electron-donor ferrocytochrome c to the four-electron acceptor dioxygen, is associated with a large degree of structural complexity. It contains four redox centers, two copper ions and two heroes, it is composed of seven dissimilar subunits and requires lipid for its activity. It participates in the coupling of the redox process to proton translocation, directed outwards in mitochondria. The resulting proton electrochemical gradient drives A T P synthesis and other ion fluxes. The enzyme can be isolated and purified by a number of techniques, which require detergents, such as cholate, deoxycholate or Triton x'2"3. After isolation, these enzyme preparations have a heme a content of 10-14 nmol and a copper content of 11-12 ng-atoms per mg of protein. T h e y also have associated, depending upon the isolation conditions, amounts of phospholipids, varying from 0.01-0.5 nmol/mg enzyme and,

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

169'

Table 1 (mol cytc/sec/mol heme) (nmol/mg protein) (ng atom/mg protein) (rag/ragprotein)

Activity Heine a Copper Phospholipid

100-300 10-14 11-13 0.01-0.5

Absorbance maxima (nm)

a, reduced ,/, reduced a, oxidized ~/, oxidized

604 445 598 417

Redox potentials (mV)

Heme a Heme a Cu Cu

(low) (high) (low) (high)

210-230 340-370 240-280 340

when 4"5 assayed under the appropriate conditions 6, can catalyze the oxidation of 100300 tool of ferrocytochrome c per second per mole of enzyme. Electron microscopy has shown that cytochrome c oxidase forms twodimensional crystalline arrays in which the enzyme complexes are ordered in a lipid milieu 7. The crystals were found to be collapsed vesicles. These crystalline structures stained with uranyl acetate were examined by electron microscopy and tilted pictures were taken. Subsequently, they underwent optical-diffraction and Fourier-transform analysis. T h e image of the cytochrome c-oxidase molecules, as obtained by the technique described, indicates that the enzyme complex is inserted asymmetrically in the membrane. Cytochrome c oxidase protrudes as much as 6 0 - 7 0 / ~ out of the bilayer (with mainly subunits II and III) 8 and very little at the other side. T h e vesicular crystals have the cytochrome c-binding site not accessible from the medium, indicating that their configuration

is inverted with respect to that in the mitochondrial membrane. It can thus be inferred that cytochrome c oxidase spans the mitochondrial membrane with a large portion protruding outside and a small part inside the matrix space 9. The resolution of the system was not such that the disposition of single subunits could be determined, although preliminary results obtained in this direction suggest that three large subunits of the enzyme span the entire thickness of the m e m b r a n e a° and that the oxidase molecules contain segments of a-helix normal to the m e m b r a n e plane 1~. The enzyme molecules, in their monomeric form, appear roughly cylindrical, approximately 5 5 A in diameter and 8 0 - 9 0 / ~ in length, with the long axis spanning the lipid bilayer and most of the protein in an aqueous phase extending from one surface of the membrane. S u b u n i t structure o f cytochrome c oxidase

T h e apparent molecular weights of the cytochrome c oxidase polypeptides from different sources are compared in Table 2. The analogies in the subunit composition are evident from their numbers and molecular weights. In the beef heart enzyme, three distinct components, called a, b and c, are also often present lz although they can be digested by trypsin without loss of enzymatic activity 13. These contaminants are seen in all preparations but usually in small and variable amounts. They may be responsible for some confusion in the nomenclature of the major polypeptides 14"15. Another major source of complications in the studies of the subunit structure of cytochrome c oxidase concerns the separation of bands II and III which can be achieved with high amounts of SDS or by polyacrylamide gel electrophoresis in

Table 2 Subunit composition of cytochrome c oxidase Subunit No. I II III IV V VI VII 170

Beef-heart21 35.400 24.100 21.000 16.800 12.400 8.200 4.400

Rat-liverz2 34.000 26.800 23.700 17.000 12.500 9.500 3.600

S. cerevisiae23

40.000 33.000 22.000 14.500 12.700 12.700 4.600

N . c r a s s a a4

41.000 28.500 21.000 16.000 14.000 11.500 10.000

the presence of urea. Despite this complication, identification of subunit III is facilitated in the beef heart enzyme by the fact that it can be labeled, rather specifically by laC-Nethylmaleimide 16, while in the enzyme from S. cerevisiae, subunit II is specifically labeled 17. A further technical problem is the interchange of migration position of subunits V and VI in Weber-Osborn gels ~8 as opposed to Shwank-Munkres gels ~9'2°. Subunit VII can appear as three bands which may indicate the presence of three slightly different versions of the same polypeptide. Unlike subunit VII the other subunits appear to be in equimolar amounts 25,26. The preparative separation of the different subunits of cytochrome c oxidase has been obtained using gel filtration Sephadex-200 or Biogel P 60 in the presence of 2% SDS 9'15'27"28. The aminoacid composition determined for the isolated polypeptides of the heart enzyme indicates that subunits I, II and IlI are much less polar that the rest of the enzyme, having a content of polar aminoacids of 35.5, 46.7 and 39.9% respectively. The smaller subunits have an average content of polar aminoacids of 48.2%. The N . crassa and S. cerevisiae enzymes have a similar distribution of polar and nonpolar aminoacids among the different subunits14A5.23,24. In both Neurospora and yeast enzymes the three large molecular-weight subunits are synthesized by the mitochondrion, while the remaining polypeptides are synthesized in the cytoplasm 23,29. T h e location a n d orientation o f heroes a n d coppers

In order to establish with which of the seven cytochrome c oxidase polypeptides the hemes and coppers are associated, several experimental approaches have been attempted. Since both hemes and coppers are bound non-covalently to the enzyme, either mild separation conditions or harsher conditions following cross-linking of the redox centers may be employed. The latter approach 3° has taken advantage of the formation of a Schiff-base at high pH between the heme and the protein, which could be stabilised after reduction with borohydride. If the pH did not alter the binding of the hemes the heine binding subunit(s) should be in the low molecular-

weight range (11,500), although it should be mentioned that the pH treatment produced an irreversible inhibition of the enzymatic activity. The possibility of separating the heme- and copper-binding subunits from the complex by mild treatments has proven complicated, and the results appear conflicting. Yu et al. 31 indicated subunits I and V as the heme-binding subunits. GtrrrgiDOE et al. 32 found the heme associated with polypeptides I and III and copper with V and VII. TANAKA et al. 33 found copper specifically associated with subunit III while TZAGALO~ and MACLENNAN34 separated a polypeptide corresponding to subunit II which contained two copper atoms per molecule_ (cf. 32) Busz et al. 35 have sequenced subunits lI and VI. By analogy with the sequences of copper or heine proteins, they concluded that subunit II has a sequence typical of a copper protein and VI of a c-type cytochrome. It is obvious that the conflicting results do not allow an identification of the subunits of the oxidase bearing the redox group (cf. Table 3). Calculations of the distance between porphyrin cytochrome c and heine a, using fluorescence energy transfer as a spectroscopic ruler, have shown that the distance between cytochrome c (bound covalently to subunit III) and the heme a was 25 ~&36. Such a distance is not compatible with subunit III being the binding site for the heine. Similar data were obtained in Chance's group 37. Although the location of heme and copper has not been possible yet, data have been obtained concerning the orientation of the heme with respect to the plane of the membrane. Using electron spin resonance spectroscopy in oriented multilayers, BLASIE et al. 38 found that the heme normal lies in the plane of the membrane and the y-axis of the heme makes an angle of 30 ° with the membrane normal. Polarized optical absorption was used by BLAS[E Table 3 Location of copper and heme in cytochrorne c oxidase subunits accordingto differentstudies. Subunit

I

Heme a Copper

31, 32

II

III

IV

V

VI

VII

32 33

27

31 32

30, 35

34, 35

27 32 171

Table 4 Subunit

I

II

III

IV

V

VI

VII

C M M--C -

I I I +

C M M--C -

0 0 +

H H +

Hydrophilic labeling Authors

EYrAN et al. 42 LUDWm and C~U'ALDIlz CHAN and TRACY2°

Probe DABS DABS antibodies

I I I

Agreement

C C(M) C +

+ I = i n t r a m e m b r a n o u s (inferred),

Lipophilic labeling BISSON et al. 43 CERLETI'I and SCHATZ44 Agreement

azidophospholipid iodoaphtylazide

H = high,

C C(M) C +

C = cytosolic side,

H H +

Me = me di um,

Me Me + L = low,

M M M +

M = ma t ri x side.

H H +

L 0 T

0 0 +

0 = zero,

et al. 39 giving essentially similar results. From

et al. 42 most probably migrated in an inverted

photo-induced linear dichroism studies of the absorption changes resulting from photolysis of the complex between a3 of the cytochrome c oxidase and CO, JUNGE and D E V A U L T 4° c o n c l u d e d that cytochrome c oxidase carried out only a limited rotation around a single axis coinciding with the symmetry axis of heine a.

manner in their gel system. It appears from Table 4 that, provided a common numbering system is used, the topology of the three large molecular-weight subunits meets with rather general agreement. Studies using hydrophilic probes showed that subunits II and III were exposed, at least in part, on the cytosolic side. The labeling studies of Lvowm and CAPALDI~2 suggest that in addition they may cross the bilayer and be labeled from the matrix side, although to a smaller extent. The lack of labeling of subunit I by the hydrophilic probes suggested that this subunit was buried in the membrane. This conclusion has been supported by more direct data obtained from studies with hydrophobic probes 43'44. With this type of label both subunits I and III were highly labeled. Subunit II was also labeled although slightly less. The direct correlation of these data with the hydrophobicity of these three higher molecular-weight subunits is of interest a5'25. The use of azido phospholipids having the active group near the polar or in the nonpolar moiety has also permitted the positions of subunit I as "buried" and those of II and III as inserted in the membrane bilayer43 to be verified. The experimental findings concerning the three high molecular-weight subunits are compatible with the model of Figure 1, which also takes into consideration the findings of HENDERSON et aL 7 and PREY et al. 8. Here, subunit I is in the middle of the bilayer and subunit III is largely buried but is also in part exposed to the water phase at the cytosolic side. Subunit II which is

Topology of cytochrome c-oxidase subunits

The approach employed to explore the arrangement of cytochrome c oxidase polypeptides with respect to the membrane has been essentially to use water-soluble probes to label exposed subunits and lipid-soluble probes to label buried subunits. When cytochrome c oxidase is studied in mitochondria or in sealed, inverted mitochondrial fragments, the labelling of the enzyme by water-soluble, non-penetrating labels is different, indicating asymmetric insertion of the enzyme in the membrane. This is consistent with the earlier observation that mitochondria, but not inverted fragments can oxidise cytoehrome c 41. The reagent which has been employed most extensively is p-diazonium benzene 35Ssulfonate, which is believed not to penetrate a phospholipid membrane 12'42, although antibodies, specifically raised against different subunits, have also been used 2°. It is important that the same numbering system is employed to compare the data of the different studies. The numbering system of DOWNER et al. with seven subunits21 is adopted here. As mentioned by CHAN and TRACYz°, subunits V and VI of E'CrAN 172

I[ CYTOSOL

-

:!::1

.........

.

.

.

.

.

.

.

.

, .....

:e:x A.E

MATRIX

Fig. 1. Schematic representation of the organisation of cytochrome c oxidase in the mitochondrial membrane.

largely exposed also interacts substantially with the hydrophobic moiety of the bilayer. The situation with the low molecular-weight subunits appears to be more complicated. Subunit IV is clearly located at the matrix side, with a segment penetrating into the bilayer and is labeled by superficial azido phospholipid probes; the topology of the other subunits is still not yet determined with precision. Subunit V has been labeled from the cytosolic and matrix sides but not with hydrophobic probes. Subunit VI is not labeled by either hydrophilic or hydrophobic probes. It is possible that this subunit, which is hydrophilic in nature, is located superficially. The presence of covering polypeptides, however, may prevent its hydrophilic labeling. Subunit VII is also an interesting case. Despite its hydrophilic nature it is labeled to a very large extent by hydrophobic probes, though it is also labeled by hydrophilic ones, from both the cytosolic and matrix sides. A possible explanation for this may come from the finding of BvsE et al. 45, i.e. that, in "subunit VII" three different N-terminal end groups can be identified, consistent with the possibility that this subunit is present in three different versions in the mitochondrial membrane and that each version may be located at a different position. The labeling approach has yielded useful and precise information on the topology of cytochrome c oxidase subunits. However, a word, at least, of caution should be expressed. Chemical labeling is group specific and may not necessarily give a thorough quantitative picture of the

exposed subunits. In this respect, azido derivatives are preferable, either in the form of non-polar or polar compounds, since they have low group specificity. Lack of labeling by polar labels does not imply necessarily interaction of a subunit with the hydrophobic part of the membrane and, vice-versa, lack of labeling with hydrophobic probes may not mean exposure to the water phase. Masking polypeptides and protein-protein interactions may be responsible in a number of cases for lack of labeling. The information obtained with hydrophobic labels and with site directed polar compounds described above, is summarized in the scheme of Figure 1, although only the data which are in agreement have been utilized. The interaction of cytochrome c-oxidase subunits with each other and with cytochrome c Cross-linking reagents and spectroscopic measurements have been useful in elucidating some of the interactions between subunits in the cytochrome c-oxidase complex, although the situation is not completely clear as yet. BRIGGS and CAPALD146have shown, by using cleavable cross-linking reagents and two-dimensional ge]l electrophoresis, that subunit V could cross-link with subunits I, II, III and VII, and subunit IV with subunits VI and VII, suggesting nearneighbour relationships among the first and second group of subunits. DOCKTER et al. 36 have measured, using fluorescence spectroscopy, that the distance between subunit II and heme a is 52 ~ , indicating that the heme-binding subunit is not in contact with subunit II. The interaction of cytochrome c with cytochrome c oxidase occurs at the cytosolic side of the mitochondrial membrane and several studies have been concerned with the determination of its binding site. B~ncIJ~l~n et al. 17, using yeast cytochrome c oxidase and iso-l-cytochrome c activated with 5,5'-dithiobis (2-nitrobenzoate), succeeded in cross-linking cytochrome c specifically to subunit III of the enzyme. This result indicated that subunit III of cytochrome c oxidase contains or forms part of the cytochrome c binding site. The latter indication was supported by BissoN et al. 47 who used aryl azido cytochrome c derivatives at residues 22 and 13 to form, upon illumination, covalent complexes with the oxidase. It was found that subunit II of 173

cytochrome c oxidase cross-linked with 13arylazido-cytochrome c in either the yeast or the beef enzyme. The derivative 22-arylazido cytochrome c was unable to bind covalently to beef-heart cytochrome c oxidase but did so with the yeast enzyme. BRIGGS and CAPALD148 crosslinked cytochrome c also to subunit II in a preformed complex with the oxidase, using an 11 A bridging bifunctional reagent, dithiobissuccinimidyl propionate. It appears probable that subunit II of cytochrome c oxidase represents the binding site for cytochrome c. In fact, it is specifically crosslinked when the reactive group is on lysine 13, which is the residue in the center of the binding region at the surface of the cytochrome c molecule49. The interaction of cytochrome c oxidase with lipids

Cytochrome c oxidase can be isolated with 50-100 moles of phospholipid associated per mole of enzyme4 and this has a high activity in the absence of added lipids. Other methods lead to a lipid-depleted enzyme (1-2 moles phospholipid per mole protein) which is unable to oxidize ferrocytochrome c unless incubated in the presence of phospholipid or of a non-ionic detergent s. In the case of beef-heart cytochrome c oxidase, the main lipid which remains tightly bound, even after extraction with organic solvents, is cardiolipin, for which an essential role in the catalytic activity of the enzyme has been postulated s°. WATTS et al. sl claimed, however, that in yeast cytochrome c oxidase all endogenous lipids can be substituted with dimerystoyl phosphatidylcholine without loss of enzymatic activity. The importance of cardiolipin for cytochrome c-oxidase function is also indicated, however, by the fact that cardiolipin was found, at least in the beef enzyme, to remain bound to the enzyme even in the presence of a 100-fold molar excess of dimerystoyl phosphatidylcholinesa. The enzyme has a higher activitys2 in the presence of dioleylphosphatidylcholine than with dipalmytoylphosphatidylcholine or dimerystoylphosphatidylcholine, indicating the need for a fluid environment for function. No evidence for head-group specificity has been found up to now. Using spin-labeled fatty acidss3 it was also 174

concluded that two lipid populations are present in a phospholipid-cytochrome c oxidase mixture, one fluid and interpreted as being in the bilayer region, and one highly immobilized, and interpreted as boundary lipid. This latter would constitute a lipid shell separating the hydrophobic intrinsic parts of the enzyme from the adjacent bilayer, and would be characterized by a slow exchange rate with the bilayer lipid molecules (cf. 54) These conclusions were made by JosT et al. s3 but are not supported by recent studies. SEEL~G and SEELIC55 obtained no evidence for a longlived boundary layer of immobilized lipids around cytochrome c oxidase, using deuteriumand 31p-NMR experiments. Rather than an "immobilized annulus", they found that there exists, around the cytochrome c oxidase molecule, a region of more disordered lipid extending several layers beyond the protein and lipid in this region which undergoes rapid exchange with the bilayer lipid. Is cytochrome c oxidase a proton pump?

One of the most topical controversies in modern bioenergetics centres on the role played by cytochrome c oxidase in H ~ translocation by the mitochondrial respiratory chain. According to the classical statements of the chemiosmotic hypothesiss6,s7 cytochrome c oxidase forms part of the electron-carrying arm of the third loop in Mitchell's sequence of alternating hydrogen and electron carriers. Much attention has been focussed on this proposed function of vectorial electron transport for cytochrome c oxidase, both in terms of supporting ss'sg"6° and criticising61 the chemiosmotic hypothesis. In particular, during the last two years a considerable amount of interest has been directed towards the suggestion that cytochrome c oxidase itself may be a proton pump. A particularly important feature of this proposal is that it would entail that at least some of the protons extruded via the mitochondrial respiratory chain are not directly chemiosmotic, that is they do not originate in the redox process itself. It is the purpose of this part of the review to summarise the evidence both for and against an H+-translocating activity in this enzyme, including new data obtained in our laboratory, and to discuss how such a redox-linked H + pump might operate.

A. Investigations of H ÷ movements catalysed by cytochrome c oxidase 1. Experiments with intact mitochondria The first demonstrations of a possible H +translocating activity in cytochrome c oxidase were reported by WIKSTROMand co-workers 62-64. Their experiments with mitochondria were carried out in the presence of antimycin and rotenone, to inhibit the redox activity of the respiratory chain prior to cytochrome c oxidase, and of oligomycin and N-ethylmateimide to prevent re-uptake of extruded H +, along or accompanied by endogenous phosphate. Under these conditions, using a KC1 medium, they found that K4(Fe(CN)6) which feeds electrons to cytochrome c, induced an acidification of the external medium followed by a monotonous alkalinisation. This transient acidification was dependent on the presence of valinomycin and was abolished by making the membrane permeable to H ÷ using a protonophore. Comparisons of the initial rates of ferrocyanide oxidation and H ÷ appearance showed a stoichiometry of one H ÷ appearing/electron equivalent. These observations are not explicable by the chemiosmotic hypothesis and instead they were attributed to transmembrane proton translocation by cytochrome c oxidase, concomitantly with cytochrome c oxidation. Thus, protons were ejected and then forced back into the mitochondria by the At2H formed as a result of H + translocation and intramitochondrial H + consumption in 02 reduction. In earlier experiments M~ITCHELLand MOYLE65'66 had seen no such H + appearance under essentially similar experimental conditions. These experiments had been carried out, however, in the absence of NEM which was essential for detection of the extruded H +, which otherwise underwent reuptake via the H+/phosphate symporter 62"63. It was argued 62'63"64 that this explained the failure to detect the H + extruded via cytochrome c oxidase and this rationale was strengthened particularly by the recent demonstrations 67"68'69'7° of the underestimation of the "H+/site '' ratio in the respiratory chain during activity of the H+/phosphate translocator. The fact that electron influx is believed to occur on reduction of 02 via cytochrome c oxidase in mitochondria meant that the require-

ment of the acidification for K ÷ and valinomycin did not provide strong evidence for the electrogenicity of the proposed H + translocation. This evidence was presented, however, in a later report 71 where it was shown that the ferrocyanide-induced H + efttux from mitochondria described above was accompanied by the uptake of 1Ca ÷+ ion per electron equivalent, allowing for 1 charge each to compensate for electron influx and H + extrusion. These studies were supported by the observation by SIGEL and CARAFOL172 of --3.5K + ions taken up per 2 electrons by mitochondria respiring in the presence of ascorbate and TMPD, another system which feeds electrons to cytochrome c. The experiments of WIKSTROM and co-workers were criticised strongly by MOYLE and MITCHELL7 3 who examined ferrocyanide-induced proton expulsion from mitochondria under a variety of conditions, including those described above. They also detected H + extrusion but this was always accompanied by a reduction of oxygen in excess of that which could be accounted for by ferrocyanide oxidation. This led them to propose that the extruded protons observed by them, and by WIKSTRt3Mand coworkers, came from a hydrogenated reductant in the respiratory chain which was oxidised by the ferricyanide formed on ferrocyanide oxidation, via a transmembrane Q/QH2 couple, the electrons passing to 02 via the Rieske ironsulphur protein, cytochromes ca, c and c oxidase, thus bypassing the antimycin block. It was suggested that the proton-motive system here might be N A D H dehydrogenase, the hydrogenated reductant being NADH, and that this was stimulated in some way by NEM. The above criticisms were countered by WIKSTRt3M and KRABTM who reported that in their experiments with uncoupled mitochondria, the rates of ferrocyanide oxidation and oxygen reduction were identical within experimental error, though the ratio of H ÷ consumed per O reduced sometimes fell as much as 28% short of the predicted 2. They found, however, that there was no correlation between this "shortfall" effect and the ferrocyanide-induced H ÷ extrusion which they observed. It should also be mentioned that, in earlier experiments, Mrrci-mli and MOYLE75 had seen a small ferrocytochrome c-induced extramitochondrial H ÷ appearance. They also observed 175

an extramitochondrial H + appearance of - 1 . 5 H + / 2 e - linked to T M P D oxidation in the presence of antimycin and rotenone. T h e y accounted for this as H + release from T M P D H 2 although other studies have shown that no such H + release O c c u r s 76 and the concentration of oxidised T M P D was probably too low to allow a bypass of the antimycin block 77. It seems more reasonable therefore, to propose that the protons which appeared were the result of T M P D induced H + translocation via cytochrome c oxidase, as observed by WIKSTROM and KRAB TM and S~GEL and CARAFOL172, but with endogenous Ca ++ providing charge neutralisation. A further objection to H + pumping via cytochrome c oxidase has been raised on the grounds that D A D H 2 induces the appearance of only 2 external H + when added to mitochondria in the presence of antimycin, r o t e n o n e and N E M 78. It was argued that these can be accounted for by H + released on D A D H 2 oxidation, thus indicating no H + translocated by the oxidase. These observations have been criticised 79 on the grounds that E G T A was present chelating endogenous Ca + and that only 1 mM-K+ was added, thus the cation influx essential for detection of the extruded H + may have been insufficient, although this argument has been countered 1°3. In addition, objections have been made to the use of D A D H 2 as a reduced substrate in this kind of experiment 77"79 but see 78. W e conclude that, whilst some of the above observations provide a strong indication that cytochrome c oxidase may act as a H ÷ pump in intact mitochondria, the considerable amount of contradictory data remains disturbing. W e feel that a more clear-cut case is provided by experiments with sub-mitochondrial particles and reconstituted vesicles, as discussed below.

2. Experiments with sub-mitochondrial particles

WIKSXR6M and SAAR163reported that on addition of T M P D to sonicated submitochondrial particles in the presence of internal K +, valinomycin, N-ethylmaleimide, oligomycin and potassium ascorbate, H ÷ were lost from the external medium and simultaneously there was acidification of the interior of the sub-mitochondrial particles. They concluded that, after correction for H + consumed in O2 176

reduction, 2 H + were taken up via a proton pump associated with cytochrome c oxidase per 2 electrons fed to cytochrome c. Again, these observations are not compatible with the chemiosmotic hypothesis, whereby T M P D , a m e m b r a n e - p e r m e a n t electron donor to cytochrome c, which is on the inside of the inverted sub-mitochondrial particles, should simply reduce 0 2 via cytochrome c oxidase, with a subsequent external consumption of 1H+/2e - as the only p H change, and with no accompanying H ÷ transport. In addition, owing to the production of the permeant Wurster's blue cation inside the particles there should be a discharge of the electrical potential formed by electron transport. Thus, in a well-buffered system there should also be no TMPD-induced At2H formation if the chemiosmotic view of cytochrome c oxidase is applied. In support of the chemiosmotic function, HAUSKA et aL so have reported that T M P D only supports an antimycin-sensitive ATP-synthesis and that this could be accounted for as a consequence of reduction by T M P D of cytochrome b sa. T h e y concluded therefore that cytochrome c oxidase could not act as an energy-coupling site. As argued by WIKSTR6M71 however, a considerable P : O ratio was obtained by HAUSY-,Aet aL so in the presence of sufficient antimycin to inhibit coupling site II completely, and the larger inhibitory effects at higher antimycin concentrations may have reflected the well-established uncoupling action of this substance. In support of the proposals of WIKSTROM and co-workers, two groups have reported an ascorbate/TMPD generated A g n (or its components) in sub-mitochondrial particles. GRIN~US et al. 82 observed ascorbate/TMPD-induced uptake of a permeant anion in sub-mitochondrial particles inhibited by antimycin, rotertone and oligomycin and this was abolished by KCN. A transmembrane, electrical potential of 90 mV generated by ascorbate and T M P D was measured by SORGATO et al. 83 in sub-mitochondrial particles inhibited with either antimycin or 2heptyl-4-hydroxyquinoline-N-oxide though they could detect no ApH. In a later report 84, however, these workers demonstrated a chemical proton gradient of 85 mV and a Aqj of 100 mV using the above system in a medium containing 50 mM-KC1. Presumably the ApH measured in the latter case resulted from C1-

entry allowing increased H + influx. These quantitative protonic potential measurements were carried out in the steady state using a flowdialysis technique. They provide, therefore, valuable complementary evidence for the other experiments favoring a cytochrome c oxidase H + pump, which rely largely on measurements of "bursts" of H + movement. Furthermore, their measurement of an internal acidification of the submitochondrial particles is particularly important considering the possible criticisms of the use by WIKSTROM and SA-A-R163of neutral red as an indicator of internal p H 85"s6. 3. Experiments with reconstituted cytochrome c oxidase vesicles The most simple and well-defined system for studying ion movements associated with cytochrome c oxidase is provided by artificial lipid vesicles prepared in the presence of the purified enzyme. By forming such vesicles in the presence of cholate and then dialysing away the detergent in the presence of cytochrome c oxidase, the enzyme molecules are incorporated into the vesicular membrane, probably with all their active sites facing outwards 16. In this way, a vesicular system of known enzyme and lipid composition and exactly defined internal and external medium can be formed. This is of great advantage, especially when carrying out the kind of pulse measurements described here, where even small amounts of unconsidered substances might cause large artefacts. WmSTR6M and co-workers 63"87 showed that addition of ferrocytochrome c to suspensions of reconstituted cytochrome c oxidase vesicles in the presence of valinomycin and in a KCI medium, led to the appearance of H + in the external medium. The external acidification did not occur in the presence of a p r o t o n o p h o r e or in the absence of valinomycin. Furthermore, following decay of the ferrocytochrome cinduced acid pulse, owing to the back-flux of H + to the vesicular interior where protons are consumed in the reduction of 02, the final stoichiometry of H + consumption was 1 per ferrocytochrome c oxidised, as expected from the equation:

4 ferrocytochrome c + 4 H + + 02-"-> 4 ferricytochrome c + 2 H 2 0 This led to the conclusion that the protons

appearing externally were the result of true translocation across the vesicular membranes via the p r o t o n pump associated with cytochrome c oxidase. These results were confirmed by CASEY et al. 88 but they contradicted the earlier conclusions of HINKLE and co-workers s9"9°. These workers found with reconstituted vesicles that in the presence of 1,4-naphthoquinone-2sulphonate, pulses of 0 2 caused the appearance of approximately 2 H ÷ per O atom reduced, which could be accounted for by the protons released by the reduced hydrogen carrier and indicated therefore no H ÷ translocation. In these experiments, however, there was a rapid decay of the H ÷ pulse, leading to the possible underestimation of the number of protons appearing extravesicularly per electron. This type of criticism also applies to their observation of only 1K + entering the vesicles per electron passing to cytochrome c oxidase, implying that the enzyme only performed electron transport. In experiments with ferrocytochrome c as the reduced substrate, however, HINKLE89 did observe an acidification remarkably similar to that observed by WIKSTROM and SAAR163, although in the former case the final ratio of H + consumed per electron equivalent was less than one indicating a possible net acid release. It is of great importance for the validity of the cytochrome c oxidase H + pump to establish that any ferrocytochrome c-induced proton appearance in the external phase is not a netacidification artefact caused by the addition, binding or oxidation of ferrocytochrome c, as may occur with intact mitochondria 73"78 but see 7v. A strong argument against this is that in the presence of CCCP 1H + is consumed per electron equivalent 87'8s. If net acidification were occurring this ratio should be less than 1. A net acidification artefact caused by ferrocytochrome c addition is further excluded by the observation that the H + appearance can be induced by the addition of Oz instead of ferrocytochrome c s7 and that the H + appearance is inhibited by azide and cyanide, two well-established inhibitors of cytochrome c oxidase (see Fig. 2). MOYLE and MITCHELL 78 have reported that, in mitochondria, a net H + release occurs simply as a result of oxidation of cytochrome c. They have supported this with the demonstration that the acidification can be induced by ferricyanide in an anaerobic system in the presence of 177

i2

INHIBITION OF CYTOCHROMEc OXIDASEBY CYANIDE INH[BITION (%) I00 90 80

PROTONTRANSLOCATION

70

X~OXIDATIONOF CYTOCHROMEc

50

30 20 10

56

160

,00

300 KCNpM

200

500

(a) INHIBITION OF CYIOCHROMEc OXIDASEBY AZIDE

PROTO ANSLOCATION 100 II'ttlBIIION (1)

A

•

90 80

•

OXIDATIONOF CYTOCHROMEc

70 60 50 40 30 20 i0 L

NAN3 ~°I

(b Fig. 2. Inhibition of cytochrome c oxidation and H+-translocation by cytochrome c-oxidase vesicles using cyanide and azide. (a) 0 . 2 m l of a suspension of cytochrome c oxidase vesicles (0.12 nmol of the enzyme) in 79 mM sucrose, 30 mM-KC1, 1 mM-Hepes, p H 7.4 was incubated for 15 hours at 4 °C with KCN at the concentrations indicated, thus providing optimal Conditions for binding of cyanide to the oxidised enzyme 91. Oxidative and H+-translocating activities of the reconstituted vesicles were then determined as described elsewhere ss. (b) 50 p,1 of a suspension of cytochrome c oxidase vesicles (0.12 nmol of the enzyme) was incubated at 12.5 °C, for 5 mins in 1.3 ml of 75 raM-choline chloride, 25 mM-KC1, 50 txM-Phenol Red, containing 2.5 txl of 0.2 mM-valinomycin, p H 7.4 (for the H+-pump measurements) or 1 ml of 150 mM-KC1, 1 mM-Hepes, containing 2.5 Vd of 0.2 mi-valinomycin, p H 7.4 (for the oxidation-rate measurements) in the presence of NaN 3 at the concentration indicated. Oxidative and H+-translocating activities of the reconstituted vesicles were then determined as described elsewhere 88,

178

Table 5 Ferrocytochrome c-induced H + extrusion from cytochrome c oxidase vesicles in the presence of varying concentrations of MgSO 4

MgSO 4 (mM) 0 10 20 30 40

H + extruded (nmol) 0.44 0.47 0.46 0.61 0.62

The H+-translocating activity of cytochrome c oxidase vesicles was determined spectrophotometrically as described elsewhere (89), except that MgSO 4 was added, prior to ferrocytochrome c, at the concentrations indicated.

ferrocytochrome c, C N - and FCCP. As shown in Figure 2, in the presence of 2 mM-NaN3 both the oxidative activity of cytochrome c oxidase and the ferrocytochrome c-induced acidification in cytochrome c oxidase vesicles were almost totally inhibited. U n d e r these conditions, addition of 30/xg-potassiurn ferricyanide, whilst causing oxidation of the ferrocytochrome c by-passing the inactivated oxidase, induced no concomitant p H changes. This excludes the possibility of H ÷ release linked to simple oxidation of ferrocytochrome c in our system. A further indication that the artefact of MITCHELL and MOYLE is specific to the mitochondrial system is that MgSO4, which inhibits their artefact totally at a concentration of 10 m M 7s, did not inhibit the ferrocytochrome c-induced proton pulse in reconstituted vesicles at concentrations up to 40 mM (see Table 5); in fact, the proton pulse was increased in size at higher Mg ÷ concentrations, as observed in mitochondria by WIKSTR6M and KRABv4. W e conclude that the ferrocytochrome c-induced proton pulse in reconstituted vesicles does indeed represent an outwardly-directed translocation of H + across the vesicular membranes. The stoichiometry of H + ejected from the reconstituted vesicles per electron equivalent was found by KRAB and WIKSTR6Ms7 to be 1 on comparing initial rates of ferrocytochrome c oxidation and H + appearance. Their maximal H + pulses, however, indicated - 0 . 7 H + ejected per electron after extrapolation to zero time. In single turnover experiments, a proton expulsion 12A

occurs which does not decay significantly 88 and therefore does not require correction by back extrapolation, presumably as the AtiH produced is very small. U n d e r these conditions, stoichiometries of H ÷ extruded per electron equivalent as high as 0.9 have been obtained as. The measured ratio of H + extruded per electron decreased with increasing turnovers of the oxidase 88'92, and this was due presumably to increased ACH build-up, more rapid H ÷ back-flux and increasing underestimation of the H + pulse. Indeed, on extrapolation of the ratio to zero turnovers, and thus zero AtiH, a value of 0.9H+/electron was again obtained. It seems reasonable therefore to conclude that 0 . 7 - 1 H ÷ is expelled per electron. As the minimum reaction catalysed by cytochrome c oxidase is the reduction of a molecule of 0 2 with the transfer of 4 electrons, this would correspond to the more meaningful ratio of - 3 - 4 protons expelled per 0 2 molecule reduced.

B. Measurements of stoichiometries of H + ejected per coupling site in the mitochondrial respiratory chain Indirect evidence concerning an H +translocating activity for cytochrome c oxidase mgy be obtained from measurements of the number of protons ejected per electron pair passrag through the third chemiosmotic loop. According to the chemiosmotic hypothesis, it is essential that this ratio be 2 for all 3 loops or "energy-coupling sites". It was first proposed that the third loop is composed of ubiquinone as hydrogen carrier, with cytochrome c and cytochrome c oxidase acting as electron carriers 56"57. This was modified later 93 so that loops 2 and 3 were combined to form the "Q cycle", with the combined loops ejecting 4 H + per 2 electrons passing to oxygen. Using O2-pulse experiments, a ratio of 2H ÷ ejected per 2 electrons per coupling site was indeed measured by MITCHELL and MOYLE 94'9s. Whilst these measurements have been supported by other laboratories, (see 96 for review), the question of the H + per site stoichiometry has had to be reviewed in the light of thermodynamic considerations 97"9s. In the recent series of publications 67-7°, BRAND and co-workers have suggested that the true stoichiometry of H + ejected per site has been underestimated in the past, owing to lack of correction for uptake of 179

some of the extruded H + via the H+/phosphate symporter, concomitantly with the uptake of endogenous phosphate by the mitochondria. Strong experimental support for these criticisms has been provided by the observations that the H+/2 electron stoichiometry is increased by washing away leaked phosphate before the measurement, or by inhibiting phosphate uptake, e.g. using NEM a specific inhibitor of the translocator. By avoiding the re-uptake of phosphate, these workers have observed, under a variety of conditions, 3-4 protons ejected per 2 electrons per site, This ratio would deviate from the chemiosmotic view and, applied to site 3, would indicate that an extra 1 or 2H ÷ must be ejected per 2 electrons by some auxiliary mechanism. A novel treatment of H+/site stoichiometries, recently put forward by BRAND et al. 99 would modify this ratio for sites 1 and 2 but retain the charge separation per 2 electrons for the span cytochrome c to 02 as 4, thus indicating 2H ÷ ejected by cytochrome c oxidase. The most recent evaluation, by ALEXANDRE eta/. ~°°, for H + ejected at site 3 indicates that all 4 protons are ejected via cytochrome c oxidase, although this would entail an even greater H + extrusion via this enzyme than those measured elsewhere. MITCHELL and MOYLE7sAm reaffirmed the stoichiometry of 2H ÷ extruded per 2 electrons at site 3 using DADH2 as substrate and showed that NEM had no effect on this, arguing that the apparent increase in the H + per site ratio in the presence of NEM was due to stimulation by NEM of NADPH-linked respiration. These experiments were criticised by WIKSTROM and KRAB, however, as mentioned in section A. A more detailed critique of the experiments indicating H+/2 electron ratios above 2 has recently been presented l°a. Further stoichiometric measurements have been made favouring the chemiosmotic view of site 3 by PAPA et al. 1°3. They showed that 2H ÷ were ejected on the passage of electrons from duroquinol to 02 and that this was all associated with the segment before cytochrome c. WIKSTR()M and KRAB TM, using duroquinol as substrate, however, have measured a ratio of 6H + ejected per 2 electrons passing through the span from ubiquinol to 02, allowing for 2H + extruded via cytochrome c oxidase. Oa-pulse measurements of H + translocation in 180

mitochondria inhibited by antimycin and 2heptyl-4-hydroxyquinoline-y-oxideperformed by PAPA et al. 1°3, also indicated no H + translocation by cytochrome c oxidase. These latter experiments were criticised71, however, on the grounds that these workers had used concentrations of antimycin and 2-heptyl-4hydroxyquinoline-N-oxide well in excess of those required to cause uncoupling, and that consequently some or all of the cytochrome c oxidase-mediated H + translocation was not detected by them.

C. Afx~-dependent changes in the optical absorbance spectrum of cytochrome c oxidase Whilst extensive studies have been carried out on energy-linked, spectral changes in cytochrome c oxidase (see 105 for review), of particular interest with regard to a possible H+-translocating activity for this enzyme is the shift occurring between 417 and 438 nm. An ATP-induced shift in this region was proposed to reflect an energy-linked conformational change affecting the ferric haem of the enzyme 1°6"1°7. This was studied in more detail by WIKSTROMand co-workers 1°s-~1° who showed that a similar shift was caused by simply inducing a transmembrane AqJ. It was also shown, however, that both in mitochondria62 and in reconstituted vesicles63 the shift was quantitatively correlated with the total transmembrane A/i H and not simply the AtO component. Whilst studies using spectrophotometric potentiometric titrations have provided evidence both for 1~ and against ~lz, but see also 77 H + movements mediated by cytochrome c oxidase, we feel that this A~H-linked spectral shift provides good complementary evidence for a conformational change in the enzyme which may be linked to H ÷ translocation. The mechanism of the cytochrome c oxidase proton pump On the basis of the available evidence outlined above, we conclude that in sub-mitochondrial particles and reconstituted vesicles, cytochrome c oxidase acts as a proton pump and it is reasonable to expect, therefore, that it would also act as such in intact mitochondria, although the evidence concerning this latter system is rather equivocal at this stage. The function of cytochrome c oxidase, therefore, should not be

considered solely to be part of a direct chemiosmotic mechanism, proposed by MrrCHELL56'57 to take the form of a redox loop. Instead (or in addition), it appears that cytochrome c oxidase acts as a p r o t o n pump in a manner which is analogous to the proton-translocating adenosine triphosphatases i.e. the free energy released by the chemical reaction catalysed by the enzyme, in this case the oxidation of cytochrome c by oxygen, is indirectly or conformationally coupled to the transmembrane movement of H ÷. As pointed out by MITCHELL113, the chemiosmotic hypothesis itself is independent of the mechanism by which the redox changes and H ÷ translocation are coupled. Consequently, whilst a conformation-linked mechanism lacks, unfortunately, the simplicity and information content of the chemiosmotic loop, it need not detract from the correctness of the essential postulates of the hypothesis. It is of interest at this stage to consider the structural basis of the redox-driven H ÷ pump of cytochrome c oxidase. It has been suggested by WIKSTROM and co-workers 64 that the high molecular-weight subunits of cytochrome c oxidase may form a proton channel whereby translocation of H ÷ through the enzyme (and across the mitochondrial membrane) takes place. This proposal was based indirectly on two observations. First, that following removal of the high molecular-weight subunits from the enzyme, oxidative activity was still present and the redox centres were unaffected ~4"1.5. Second, that removal of lipid caused aggregation of the heavy subunits, as indicated by their separation pattern on SDS gel-electrophoresis, indicating a possible stable interaction between these in the hydrophobic environment of the mitochondrial m e m b r a n e 64. The latter evidence is questionable considering that delipidation of cytochrome c oxidase by centrifugation through a sucrose layer in the presence of cholate results in an enzyme which gives the well-established electrophoresis pattern ~16. More direct evidence for the involvement of individual subunits of cytochrome c oxidase in the formation of a H + channel has been obtained recently, in this laboratory. W e reported x~7 that DCCD, a well-established inhibitor of ATP-linked proton pumps a~8-12° also inhibits proton translocation by cytochrome c oxidase, both in reconstituted vesicles and in

8¢

~--~60

RECONSTITUTED VESICLES

O 40

~

.*,

z

o_ n,. ix ~u 20 z

MITOCHONDRIA

e,, D.

o

1;o 26o 3;0

4;0 ~o

// 15bo

nmoles DCCD per nrnole cytochrome c oxidase

Fig. 3. Inhibition of cytochrome c oxidase m e d i a t e d H + translocation by D C C D . For e x p e r i m e n t a l details see Ref.

117. rat-heart mitochondria (see Fig. 3). Stimulated by this finding, we have investigated the possibility that this substance binds covalently to the oxidase, as it does to one hydrophobic subunit in H+-translocating ATPases lam22. Our studies (to be reported in full elsewhere) show that radioactively-labeled D C C D binds covalently to subunit III of cytochrome c oxidase when the enzyme is incorporated into vesicles and to subunits III and IV in the free enzyme (see Fig. 4). As it has been suggested that subunit III of the enzyme spans the mitochondrial membrane 12, then the possible role of this subunit as providing the H ÷ channel in the enzyme can be seen as an important indication provided by this observation.

Concluding Remarks W e have tried to present an outline of the existing knowledge of cytochrome c oxidase which, whilst not totally comprehensive, summarises those aspects which are particularly topical at the moment, some of which we are investigating in our laboratory. It is clear that whilst our understanding of the structure and function of this enzyme is considerable, many important questions remain unanswered. We feel, therefore that the field is ideally poised for the pursuit of fruitful research. In particular, the 181

I

3001

d

d

I

II

III

IV V Vl

VII

"~ 2000

1000

~ (D

200

I

II

III

IV

VVl

VII

100:

,la

DISTANCE FROM ORIGIN

DISTANCE FROM ORIGIN

Fig. 4. Labelling of free and reconstituted cytochrome c oxidase by 14C-DCCD. Cytochrome c oxidase either suspended in 250 mM-sucrose, 50 raM-sodium phosphate buffer pH 7.4 (A) or reconstituted into vesicles (B) a8 was incubated for 15 hrs at 4 °C with 14C-DCCD (specific activity 50 mCi]mmol) at a ratio of 20 nmole DCCD per nmol enzyme. Lipid and unbound DCCD were separated from this mixture by centrifugation through 10% sucrose in the presence of cholate 116. An aliquot of the resuspended pellet underwent electrophoresis on a gel containing 12% polyacrylamide. The gel was stained using Coomassie blue and following destaining was scanned at 600 run, giving the absorbance trace shown. The gel was then sliced and the slices dialysed for 60 hrs versus 7% CHaCOOH followed by heating for 5 hrs at 80 °C with 30% H202, 1% concentrated ammonia solution and scintillation counting to give the radioactivity distribution shown.

recent studies on the application of chemical probes of the enzyme structure and on a proton-translocating function for the oxidase present new experimental approaches which we hope will lead to a more thorough understanding of this important respiratory enzyme.

Acknowledgements We thank DRS R. BISSON, J. B. CHAPPELL, H. GUTWENIGER, C. MONTECUCCO and M. THELEN for their collaboration on the original work presented here which was supported in part by the Swiss National Science Foundation (Grant No. 3.288-077), the Emil Barrel1 Stiftung and the Clark Joller Fund. We are also grateful to DRS P. MITCHELLand M. WIKSTRt)Mfor supplying articles before their publication.

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182

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