ANNUAL REVIEWS
Further
Quick links to online content Rev. Biochem. 1990. 59:569-96 Copyright © 1990 by Annual Reviews Inc. All rights reserved
Annu.
STRlfCTURE AND FUNCTION OF
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
CYTC)CHROME
c
OXIDASE
Roderick A. Capaldi Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 KEY WORDS:
respiratory chain complex, terminal oxidase, oxygen-binding enzyme, energy coupling, heme proteins.
CONTENTS INTRODUCTION .....................................................................................
569
COMPOSITIONAL STUDIES................................................... . ............. . . . . . Prosthetic Groups and Metal Content of the Enzyme . . . . . . . ......... ........ . . . . ......... Subunit Structure...... ....... . . . . ....... . ........................... ......... . . ...................
570 570 571
THE CATALYTIC CORE ..........................................................................
573
Subunit III and Proton Pumping Function. . ........... . . . . . . . . . ... . . ..... . . . . . . . . . . ... . . . . . . Arrangement of Subunits I, II, and III in the Enzyme Complex . . . . . . . ...... . . . . . . . . . ..
578 578
THE NUCLEAR-CODED SUBUNITS .............. . . . . .............. . . . .......... .............. Structural Features . . . . . . ... . . . . . . ..... . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . ... . ........ Isoforms. . . ...... ............. ....... . . . .......... . . .. ............... .... . . .......... ... . . ....... . . . .
579 579 580
AGGREGATION STATE AND OTHER GROSS STRUCTURAL FEATURES . . . . ... .
581
MECHANISM ......................................................................................... Cytochrome c Binding . .............................. . . ... . ................ . . . . . . ... ....... ...... Electron Transfer Pathway... . .... .... . . . ..... ...... ......................... ............... . . . . Conformational States Associated with Redox Activity ........ ....... ........... . ......... Proton Pumping Mechanism ........ . . . ........... . ..... ............ . . .... . ........... . . . . . . ... .
582 582 585 586 587
ROLE OF NUCLEAR-CODED SUBUNITS .................................................... Control of Enzymatic Activity . . . . . . . ...... ...... . . . ....... ....... . . . . .............. . . ...... . . . . Biogenesis of the Enzyme Complex..... ................... . ..... . . .......... . .... ...... . . . . . .
588 588 589
CONCLUSIONS AND PROSPECTS .............................................................
590
Apoproteins for the Redox Centers............................................................
573
INTRODUCTION
Few enzymes have commanded as much attention or are the subject of as much study as cytochrome c oxidase (EC 1.9.3.1). A book has been dedicated 569 0066-4154/90/0701-0569$02.00
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
570
CAPALDI
entirely to the structure and functioning of the enzyme (1), and meetings have been organized to discuss this protein alone (2). Cytochrome c oxidase is a complex metalloprotein that provides a critical function in cellular respiration in both prokaryotes and eukaryotes. The enzyme catalyzes the reaction 4H+ + 4e- + O2;;= 2 H20. Energy released in this exogonic reaction is conserved as a pH gradient and membrane potential across the membrane barrier, generated in part by H+ consumption and also by proton translocation through the protein complex. This review focuses on aartype cytochrome c oxidase, as opposed to ab- or ao-type oxidases, which are found in some bacteria (3) , and considers the structure-function rela tionships that are emerging from studies on the enzyme from a variety of organisms, including bacteria such as Paracoccus denitrificans, and eu karyotes as various as slime mold, yeast, and human. Characterization of cytochrome c oxidase from yeast has opened the way for studying the biogenesis of the enzyme. Cytochrome c oxidase in eu karyotes is encoded in two genomes. Several polypeptides are coded for on mtDNA and synthesized inside the mitochondrion, while others are coded in the nucleus and made in the cytoplasm. Genetic and molecular biological approaches are being used to examine the communication that must exist between the two genomes in regulation of the synthesis of cytochrome c oxidase. Assembly of the enzyme is also being studied, and there is consider able interest at present in the way that the cytoplasmically made subunits of cytochrome c oxidase are targeted to and enter the mitochondrion. These aspects of research on cytochrome c oxidase have been covered in several recent reviews (4--6) . Recently, medical researchers have become interested in cytochrome c oxidase, with the realization that a number of diseases of humans result from deficiencies in this enzyme. For a review of diseases involving cytochrome c oxidase, the reader is referred to articles by DiMauro et al (7) and by Kennaway et al (8). COMPOSITIONAL STUDIES Prosthetic Groups and Metal Content of the enzyme The redox centers of both prokaryotic and eukaryotic aartype cytochrome c oxidases are two heme a moieties (a and a3) and two copper atoms (CUA and CUB) (9-11) . Heme a appears to be liganded by two His residues (12, 13), CUA by two His and two Cys ( 14-- 1 6), heme a3 by one His (17, 18), and CUB by three His residues (19, 20). Heme a3 and CUB are then bridged by a shared ligand, which could be a sulfur-containing residue (21), a /-L peroxy group (10), or a chloride ion (19). Heme a differs from b-, c-, or o-type hemes in having a formyl group in
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
CYTOCHROME c OXIDASE
571
position 8 of the porphyrin ring and a long isoprenoid chain in position 2 . The formyl group of heme a (but not heme a3) is hydrogen bonded to an amino acid side chain as judged by laser Raman spectroscopy (13), possibly to a Tyr residue (22) . The distance between heme a and CUA in the protein complex has been measured ,as 8-1 0 A (23); heme a and heme a3 are 1 2- 1 6 A apart (24, 25) and heme a3 and CUB are 3� A apart ( 1 9, 2 1 ). Recent metal compositional studies indicate that there are 1 mol each of Mg and Zn in eukaryotic cytochrome C oxidases, and that Mg but not Zn is present in the baclterial enzyme (26-33) . Extended X-ray Absorption Fine Structure (EXAFS) studies suggest that the Zn atom is liganded by one His and three sulfur-containing residues, either Cys or Met residues (30) . It has been claimed that there is a third copper center (Cux) in bacterial and eukaryotic cytochrome C oxidases (26-28, 33), which is present at one mol per monomer of oxidase according to Steffens et al (28) and in one mol per mole of enzyme dimer according to Einarsdottir & Caughey (27). Li et al (3 1 ) find n o evidence for more than 2 Cu per 2 Fe, suggesting that CUx i s an a�ventitiously associated impurity. Certainly it is not a redox-active com ponent. Similarly, Zn does not appear to function in electron transfer and the role of this metal center, as well as that of Mg, remains to be defined. Subunit Structure The subunit compositions of cytochrome c oxidase from several different organisms are listed in Table 1 . Three numbering systems are presented, two currently in use for the mammalian enzyme (34, 35) and one in use for the yeast enzyme (36). Most workers are now using the nomenclature of Kaden bach et al (34) to identify the subunits of the mammalian enzyme, and this system is adopted here. The number of subunits copurifying with heme aa3 in cytochrome c oxidase preparations varies with the organism being studied. Thus the enzyme from bacterial sources contains at most three subunits, while there are as many as 13 different polypeptides in mammalian cytochrome c oxidase. The amino acid sequences of the subunits of two bacterial cytochrome c oxidases, those from Paracoccus denitrificans (37) and PS3 (38) , have been deduced after cloning the genes for these polypeptides. The amino acid sequences of subunits from several eukaryote cytochrome c oxidases have been obtained , either directly , or from DNA sequence data (e. g . 39-68) . It is possible, therefore , to identify components in common among the various oxidases. The three subunits of bacterial aartype cytochrome C oxidases are the homologues of the mitochondrially coded subunits (I, II, and III) in eu karyotes. Four nuclear-coded subunits, IV, Va, Vb, and VIc, are found in
572
CAPALDI Table 1
Subunit structure of cytochrome c oxidase in
different organisms
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
Bovine (heart)
Yeast
Pd
C
M,
M,
56993
I
56000
55000
26049
II
26678
27000
MtIII
299 1 8
III
30340
30000
IV
CIV
1 7 153
V
1 4858
Va
Cv
1 2434
VI
1 2627
Vb
1 0670
IV
1 4570
VIa
CVI ASA
VIb
AED
10068
A
B
M,
MIl II
MIll
III
94 1 8
VIc
STA
8480
VIla
6303
VIla
6234
VII
6603
VIIb
CVII IHQ
6350
VIIc
CVIII
5541
VIII
5364
VIII
C,x
4962
(A) Nomendature of Kadenbach & Merle (34). (B) Nomendature of Capaldi et al (35). (C) Nomenclature of Power ct al (36).
lower eukaryotes such as slime mold and yeast and are also present in mammals (34, 35 , 69) (IV, V, VI, and VIla of yeast). Two subunits present in yeast are absent in slime mold: these are subunits VIla and VIIc (VIla and VIII of yeast) (70) . Finally, there are four subunits in mammals not found in the lower eukaryotes: these are numbered VIa, VIb, VIIb, and VIII. There is genetic evidence that IV, Va, Vb, Vic, and VIIc are bona fide components of the cytochrome c oxidase complex. Null mutants in the genes of any of these subunits in yeast either fail to assemble cytochrome oxidase, or yield a cytochrome c oxidase with diminished activity compared to wild type (71-76) . Similar data are not available for VIla (VII of yeast) because the gene for this subunit has not been cloned. Moreover, there are as yet no genetic studies in mammals from which to decide whether VIa, VIb, VIIb, and VIII are true subunits or adventitiously associated impurities. However, these four polypeptides are isolated as part of the cytochrome c oxidase complex from several different tissues of a variety of different animals (34, 35, 77-79), and this suggests that they are bona fide subunits. Merle & Kadenbach (77) have claimed that all 1 3 subunits of the mammalian enzyme copurify in stoichiometric amount with heme (aa3) in several different isola tion procedures. This is not the experience of most laboratories (9, 80, 81). Subunits III, VIa, VIb, and VIla are readily removed by detergent treatment and are often missing in part and sometimes totally from enzyme preparations (9, 80, 82, 83) .
CYTOCHROME
c
OXIDASE
573
THE CATALYTIC CORE
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
Apoproteins for the Redox Centers The data on the composition of cytochrome c oxidase from different sources (above) h21ve important implications for the structure and function of this enzyme. They indicate that there is a minimal unit competent for both the electron transfer and proton pumping functions (self-evident from studies of the simpler bacterial cytochrome c oxidases). Furthermore, they imply that the mitochondrially coded subunits I, II, and III are the catalytic core of the enzyme in eukaryotes. Hemes and coppers can then be localized to subunits I and II, because subunit III is readily removed from both the prokaryote and eukaryote enzyme without loss of these metal centers (84, 85). Ligands for the prosthetic groups should be conserved residues. A total of eight His and two Cys residues are required to bind the four redox centers (see earlier). Subunit I contains seven conserved His but no conserved Cys as indicated by the sequence com parisons shown in Figure 1 , while subunit II has two conserved His and two conserved Cys residues. This argues for subunit II containing CUA with the rest of the redox centers in subunit I (assuming that the prosthetic groups are in individual polypeptides and do not share ligands with two or more sub units). Holm et al (86) have proposed a model for the arrangement of redox centers in subunit II in which heme a is Jiganded by His232 and His366 (numbering for the yeast subunit), heme a3 by Hisz39, and CUB by His288, His289, and His374. These authors place the redox centers within the bilayer, intercalated among a set of transmembrane helices. No experimental verification for the model of Holm et al (86) has been reported to date. The locus of CUA in subunit II has more experimental support. It has been pointed out by Steffens & Buse (65) that subunit II has a weak homology with the copper-binding regions of azurin and plastocyanin. Also Hall et al (87) have removed CUA selectively from beef heart cytochrome c oxidase by reacting th,e enzyme with p-hydroxymercuribenzoate (pHMB) and showed that the only Cys residues exposed by this treatment are in subunit II. The most likely binding site for CUA is the loop containing CYSI96, CYS200, and His204, with the remaining conserved histidine (His161) providing the fourth ligand. The view that subunit II contains CUA is not universally held. Azzi and colleagues (88, 89) have claimed to remove subunit II from Paracoccus denitrificans by protease digestion with only partial loss of activity and with CUA still bound. These workers propose that subunit II contains CUx, with CUA, CUB, and the two hemes all in subunit I. Sequence data on a cDNA for subunit II of wheat have also been used to
574
Yeast
[ref.
45]
.............. MYQRWLySTN
ML
Beef
[ref. 39] [ref. 40] [ref. 42]
.............MGINRWLFSTN
LF LF LF
[ref. 44] [ref. 43] [ref. 46] [ref. 54]
.............. MSRQWLFSTN .............MQLSRWLFSTN . . . • . . . . . . . . . MAITRWLFSTN
IF IF
. . . . MTNPVRWLFSTN MSSISVVTERWFLSTN
IF IF FT
22 22 23 27 35
IS
33
Human Mouse Dros PIs Xen Den Neu Pd PS3
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
CAPALDI
[ref. 62] [ref. 37] [ref. 38]
•
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•
•
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•
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•
•
•
•
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•
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.
.
.
MFADRWLFSTN MFINRWLFSTN
21 22 22
22 21
VF
MSAQISDSIEEKRGFFTRWFMSTN MSTIARKKGVGAVLWDYLTTVD
Yeast
AIFSGMAGTAMSLI
SQY .
.
•
.
.
.
.
.
.
LHGN
51
Beef
GAWAGMVGTALSLL
TLL •
.
.
.
.
.
.
.
.
GDDQ
Human Mouse Dros PIs
GAWAGVLGTALSLL GAWAGMVGTALSIL GAWAGMVGTSLSIL GAWAGMVGTAMSVI
GNLL.........GNDH GALL.........GDDQ GALI . • . . . . . • • GDDQ .uw�ulnuSLL.........NDDQ
52 52 52
Xen
GAWAGLVGTALSLL
Den
52 53
Pd PS3
GAIAGVMGTLFSVL ALFSGLLGTAFSVL AGLAGLISVTLTVY GGFFFLLGGLEALF
TLL.........GDDQ ����DQI . . . . . . . . • LGGN VQY.........IADN VQYMCLEGMRLVPNAH
74
NDFLV •
63
Yeast Beef Human Mouse
SQL • . FNVV IYN ..VVVT IYN.. VIVT IYN .. VIVT
Dros PIs
IYN.. VIVT IYN.. VVVT
Xen Den
IYN..VIVT HQLYNVLIT QLYN.AIIT
Neu
Neu Pd PS3
Yeast Beef Human Mouse Dros PIs Xen Den Neu Pd PS3
•
.
.
.
•
•
.
.
GG
51
52
57
UPUMDQA'T
89
P
P
89
P P
90 90 93
ur-J�"""'P P
96
\llPI.lMI�P vWr.lMtljAP \llPI.lMI�P
90 90
90
LWN.. VVVT LYNE .._ V�L�T������..�______
112 ���.R 100
L.....VESGAG M.....VEAGAG M.....VEAGAG M.....VEAGAG M ••... VENGAG G..... VESGAG G..... VEAGAS L.....VEVGSG
C
. . . . . IEGGAG LSPGGSDQPGAG
FLGGAPD . . . . .
124
125 125 125 124 125 125 128 131 152 135
CYTOCHROME
Yeast Beef Human Mouse Dros PIs Xen Den Neu
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
Pd PS3
Yeast Beef Human Mouse Dros PIs Xen Den Neu Pd PS3
Yeast
Beef
Human Mouse Dros PIs Xen
IYP LYP SYA
GNLAHAGAS GITSHSGGA GVQSHSGPS TTEA .. GYA LDSKAHHG.
��� �� 'lj ��lt�t
575
LAlFALHLTSISSLLGA LTIFSLHLAGVSSILGA LTI F SL H LAGV S S I LGA LTlFSLHLAGVSSILGA LAIFSLHLAGISSILGA LAIFSLHLAGASSlLAS TIFSLHLAGASSILAS AISSLHLSGVSSILGS AIFALHLSGVSSLLGS AIFAVHVSGATSLLGAI YTLGL lSGFGTIMGAI
I 164 I 165
I 165
I 165 I 164 I
165
I 165
I 168
I 171
190 174
204 205 205 205
TNGMTMHKLP PPAMSQYQTP PPAMT QYQTP PPAMTQYQTP STGISLDRMP
Gil.
204
Gil. 205
TPGMSFDRLP
PPAMSQYQTP GLGMTMHRSP
AG 205 208 TPGIRLHKLA 211 ��Tl� M�APGMTLFKIP 230 ���r,APGMTFMRMP�� ���__����__� 214 ITMLL
PILYQ PILYQ PILYQ PILYQH •. "'IN..·..· PILFQ PVLYQHO."W..'"· P I LYQH •. "WI
Further
Quick links to online content Rev. Biochem. 1990. 59:569-96 Copyright © 1990 by Annual Reviews Inc. All rights reserved
Annu.
STRlfCTURE AND FUNCTION OF
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
CYTC)CHROME
c
OXIDASE
Roderick A. Capaldi Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 KEY WORDS:
respiratory chain complex, terminal oxidase, oxygen-binding enzyme, energy coupling, heme proteins.
CONTENTS INTRODUCTION .....................................................................................
569
COMPOSITIONAL STUDIES................................................... . ............. . . . . . Prosthetic Groups and Metal Content of the Enzyme . . . . . . . ......... ........ . . . . ......... Subunit Structure...... ....... . . . . ....... . ........................... ......... . . ...................
570 570 571
THE CATALYTIC CORE ..........................................................................
573
Subunit III and Proton Pumping Function. . ........... . . . . . . . . . ... . . ..... . . . . . . . . . . ... . . . . . . Arrangement of Subunits I, II, and III in the Enzyme Complex . . . . . . . ...... . . . . . . . . . ..
578 578
THE NUCLEAR-CODED SUBUNITS .............. . . . . .............. . . . .......... .............. Structural Features . . . . . . ... . . . . . . ..... . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . ... . ........ Isoforms. . . ...... ............. ....... . . . .......... . . .. ............... .... . . .......... ... . . ....... . . . .
579 579 580
AGGREGATION STATE AND OTHER GROSS STRUCTURAL FEATURES . . . . ... .
581
MECHANISM ......................................................................................... Cytochrome c Binding . .............................. . . ... . ................ . . . . . . ... ....... ...... Electron Transfer Pathway... . .... .... . . . ..... ...... ......................... ............... . . . . Conformational States Associated with Redox Activity ........ ....... ........... . ......... Proton Pumping Mechanism ........ . . . ........... . ..... ............ . . .... . ........... . . . . . . ... .
582 582 585 586 587
ROLE OF NUCLEAR-CODED SUBUNITS .................................................... Control of Enzymatic Activity . . . . . . . ...... ...... . . . ....... ....... . . . . .............. . . ...... . . . . Biogenesis of the Enzyme Complex..... ................... . ..... . . .......... . .... ...... . . . . . .
588 588 589
CONCLUSIONS AND PROSPECTS .............................................................
590
Apoproteins for the Redox Centers............................................................
573
INTRODUCTION
Few enzymes have commanded as much attention or are the subject of as much study as cytochrome c oxidase (EC 1.9.3.1). A book has been dedicated 569 0066-4154/90/0701-0569$02.00
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
570
CAPALDI
entirely to the structure and functioning of the enzyme (1), and meetings have been organized to discuss this protein alone (2). Cytochrome c oxidase is a complex metalloprotein that provides a critical function in cellular respiration in both prokaryotes and eukaryotes. The enzyme catalyzes the reaction 4H+ + 4e- + O2;;= 2 H20. Energy released in this exogonic reaction is conserved as a pH gradient and membrane potential across the membrane barrier, generated in part by H+ consumption and also by proton translocation through the protein complex. This review focuses on aartype cytochrome c oxidase, as opposed to ab- or ao-type oxidases, which are found in some bacteria (3) , and considers the structure-function rela tionships that are emerging from studies on the enzyme from a variety of organisms, including bacteria such as Paracoccus denitrificans, and eu karyotes as various as slime mold, yeast, and human. Characterization of cytochrome c oxidase from yeast has opened the way for studying the biogenesis of the enzyme. Cytochrome c oxidase in eu karyotes is encoded in two genomes. Several polypeptides are coded for on mtDNA and synthesized inside the mitochondrion, while others are coded in the nucleus and made in the cytoplasm. Genetic and molecular biological approaches are being used to examine the communication that must exist between the two genomes in regulation of the synthesis of cytochrome c oxidase. Assembly of the enzyme is also being studied, and there is consider able interest at present in the way that the cytoplasmically made subunits of cytochrome c oxidase are targeted to and enter the mitochondrion. These aspects of research on cytochrome c oxidase have been covered in several recent reviews (4--6) . Recently, medical researchers have become interested in cytochrome c oxidase, with the realization that a number of diseases of humans result from deficiencies in this enzyme. For a review of diseases involving cytochrome c oxidase, the reader is referred to articles by DiMauro et al (7) and by Kennaway et al (8). COMPOSITIONAL STUDIES Prosthetic Groups and Metal Content of the enzyme The redox centers of both prokaryotic and eukaryotic aartype cytochrome c oxidases are two heme a moieties (a and a3) and two copper atoms (CUA and CUB) (9-11) . Heme a appears to be liganded by two His residues (12, 13), CUA by two His and two Cys ( 14-- 1 6), heme a3 by one His (17, 18), and CUB by three His residues (19, 20). Heme a3 and CUB are then bridged by a shared ligand, which could be a sulfur-containing residue (21), a /-L peroxy group (10), or a chloride ion (19). Heme a differs from b-, c-, or o-type hemes in having a formyl group in
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
CYTOCHROME c OXIDASE
571
position 8 of the porphyrin ring and a long isoprenoid chain in position 2 . The formyl group of heme a (but not heme a3) is hydrogen bonded to an amino acid side chain as judged by laser Raman spectroscopy (13), possibly to a Tyr residue (22) . The distance between heme a and CUA in the protein complex has been measured ,as 8-1 0 A (23); heme a and heme a3 are 1 2- 1 6 A apart (24, 25) and heme a3 and CUB are 3� A apart ( 1 9, 2 1 ). Recent metal compositional studies indicate that there are 1 mol each of Mg and Zn in eukaryotic cytochrome C oxidases, and that Mg but not Zn is present in the baclterial enzyme (26-33) . Extended X-ray Absorption Fine Structure (EXAFS) studies suggest that the Zn atom is liganded by one His and three sulfur-containing residues, either Cys or Met residues (30) . It has been claimed that there is a third copper center (Cux) in bacterial and eukaryotic cytochrome C oxidases (26-28, 33), which is present at one mol per monomer of oxidase according to Steffens et al (28) and in one mol per mole of enzyme dimer according to Einarsdottir & Caughey (27). Li et al (3 1 ) find n o evidence for more than 2 Cu per 2 Fe, suggesting that CUx i s an a�ventitiously associated impurity. Certainly it is not a redox-active com ponent. Similarly, Zn does not appear to function in electron transfer and the role of this metal center, as well as that of Mg, remains to be defined. Subunit Structure The subunit compositions of cytochrome c oxidase from several different organisms are listed in Table 1 . Three numbering systems are presented, two currently in use for the mammalian enzyme (34, 35) and one in use for the yeast enzyme (36). Most workers are now using the nomenclature of Kaden bach et al (34) to identify the subunits of the mammalian enzyme, and this system is adopted here. The number of subunits copurifying with heme aa3 in cytochrome c oxidase preparations varies with the organism being studied. Thus the enzyme from bacterial sources contains at most three subunits, while there are as many as 13 different polypeptides in mammalian cytochrome c oxidase. The amino acid sequences of the subunits of two bacterial cytochrome c oxidases, those from Paracoccus denitrificans (37) and PS3 (38) , have been deduced after cloning the genes for these polypeptides. The amino acid sequences of subunits from several eukaryote cytochrome c oxidases have been obtained , either directly , or from DNA sequence data (e. g . 39-68) . It is possible, therefore , to identify components in common among the various oxidases. The three subunits of bacterial aartype cytochrome C oxidases are the homologues of the mitochondrially coded subunits (I, II, and III) in eu karyotes. Four nuclear-coded subunits, IV, Va, Vb, and VIc, are found in
572
CAPALDI Table 1
Subunit structure of cytochrome c oxidase in
different organisms
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
Bovine (heart)
Yeast
Pd
C
M,
M,
56993
I
56000
55000
26049
II
26678
27000
MtIII
299 1 8
III
30340
30000
IV
CIV
1 7 153
V
1 4858
Va
Cv
1 2434
VI
1 2627
Vb
1 0670
IV
1 4570
VIa
CVI ASA
VIb
AED
10068
A
B
M,
MIl II
MIll
III
94 1 8
VIc
STA
8480
VIla
6303
VIla
6234
VII
6603
VIIb
CVII IHQ
6350
VIIc
CVIII
5541
VIII
5364
VIII
C,x
4962
(A) Nomendature of Kadenbach & Merle (34). (B) Nomendature of Capaldi et al (35). (C) Nomenclature of Power ct al (36).
lower eukaryotes such as slime mold and yeast and are also present in mammals (34, 35 , 69) (IV, V, VI, and VIla of yeast). Two subunits present in yeast are absent in slime mold: these are subunits VIla and VIIc (VIla and VIII of yeast) (70) . Finally, there are four subunits in mammals not found in the lower eukaryotes: these are numbered VIa, VIb, VIIb, and VIII. There is genetic evidence that IV, Va, Vb, Vic, and VIIc are bona fide components of the cytochrome c oxidase complex. Null mutants in the genes of any of these subunits in yeast either fail to assemble cytochrome oxidase, or yield a cytochrome c oxidase with diminished activity compared to wild type (71-76) . Similar data are not available for VIla (VII of yeast) because the gene for this subunit has not been cloned. Moreover, there are as yet no genetic studies in mammals from which to decide whether VIa, VIb, VIIb, and VIII are true subunits or adventitiously associated impurities. However, these four polypeptides are isolated as part of the cytochrome c oxidase complex from several different tissues of a variety of different animals (34, 35, 77-79), and this suggests that they are bona fide subunits. Merle & Kadenbach (77) have claimed that all 1 3 subunits of the mammalian enzyme copurify in stoichiometric amount with heme (aa3) in several different isola tion procedures. This is not the experience of most laboratories (9, 80, 81). Subunits III, VIa, VIb, and VIla are readily removed by detergent treatment and are often missing in part and sometimes totally from enzyme preparations (9, 80, 82, 83) .
CYTOCHROME
c
OXIDASE
573
THE CATALYTIC CORE
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
Apoproteins for the Redox Centers The data on the composition of cytochrome c oxidase from different sources (above) h21ve important implications for the structure and function of this enzyme. They indicate that there is a minimal unit competent for both the electron transfer and proton pumping functions (self-evident from studies of the simpler bacterial cytochrome c oxidases). Furthermore, they imply that the mitochondrially coded subunits I, II, and III are the catalytic core of the enzyme in eukaryotes. Hemes and coppers can then be localized to subunits I and II, because subunit III is readily removed from both the prokaryote and eukaryote enzyme without loss of these metal centers (84, 85). Ligands for the prosthetic groups should be conserved residues. A total of eight His and two Cys residues are required to bind the four redox centers (see earlier). Subunit I contains seven conserved His but no conserved Cys as indicated by the sequence com parisons shown in Figure 1 , while subunit II has two conserved His and two conserved Cys residues. This argues for subunit II containing CUA with the rest of the redox centers in subunit I (assuming that the prosthetic groups are in individual polypeptides and do not share ligands with two or more sub units). Holm et al (86) have proposed a model for the arrangement of redox centers in subunit II in which heme a is Jiganded by His232 and His366 (numbering for the yeast subunit), heme a3 by Hisz39, and CUB by His288, His289, and His374. These authors place the redox centers within the bilayer, intercalated among a set of transmembrane helices. No experimental verification for the model of Holm et al (86) has been reported to date. The locus of CUA in subunit II has more experimental support. It has been pointed out by Steffens & Buse (65) that subunit II has a weak homology with the copper-binding regions of azurin and plastocyanin. Also Hall et al (87) have removed CUA selectively from beef heart cytochrome c oxidase by reacting th,e enzyme with p-hydroxymercuribenzoate (pHMB) and showed that the only Cys residues exposed by this treatment are in subunit II. The most likely binding site for CUA is the loop containing CYSI96, CYS200, and His204, with the remaining conserved histidine (His161) providing the fourth ligand. The view that subunit II contains CUA is not universally held. Azzi and colleagues (88, 89) have claimed to remove subunit II from Paracoccus denitrificans by protease digestion with only partial loss of activity and with CUA still bound. These workers propose that subunit II contains CUx, with CUA, CUB, and the two hemes all in subunit I. Sequence data on a cDNA for subunit II of wheat have also been used to
574
Yeast
[ref.
45]
.............. MYQRWLySTN
ML
Beef
[ref. 39] [ref. 40] [ref. 42]
.............MGINRWLFSTN
LF LF LF
[ref. 44] [ref. 43] [ref. 46] [ref. 54]
.............. MSRQWLFSTN .............MQLSRWLFSTN . . . • . . . . . . . . . MAITRWLFSTN
IF IF
. . . . MTNPVRWLFSTN MSSISVVTERWFLSTN
IF IF FT
22 22 23 27 35
IS
33
Human Mouse Dros PIs Xen Den Neu Pd PS3
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
CAPALDI
[ref. 62] [ref. 37] [ref. 38]
•
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•
•
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.
•
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.
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.
.
.
•
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.
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.
•
•
•
•
.
.
.
.
.
.
.
•
.
.
.
.
MFADRWLFSTN MFINRWLFSTN
21 22 22
22 21
VF
MSAQISDSIEEKRGFFTRWFMSTN MSTIARKKGVGAVLWDYLTTVD
Yeast
AIFSGMAGTAMSLI
SQY .
.
•
.
.
.
.
.
.
LHGN
51
Beef
GAWAGMVGTALSLL
TLL •
.
.
.
.
.
.
.
.
GDDQ
Human Mouse Dros PIs
GAWAGVLGTALSLL GAWAGMVGTALSIL GAWAGMVGTSLSIL GAWAGMVGTAMSVI
GNLL.........GNDH GALL.........GDDQ GALI . • . . . . . • • GDDQ .uw�ulnuSLL.........NDDQ
52 52 52
Xen
GAWAGLVGTALSLL
Den
52 53
Pd PS3
GAIAGVMGTLFSVL ALFSGLLGTAFSVL AGLAGLISVTLTVY GGFFFLLGGLEALF
TLL.........GDDQ ����DQI . . . . . . . . • LGGN VQY.........IADN VQYMCLEGMRLVPNAH
74
NDFLV •
63
Yeast Beef Human Mouse
SQL • . FNVV IYN ..VVVT IYN.. VIVT IYN .. VIVT
Dros PIs
IYN.. VIVT IYN.. VVVT
Xen Den
IYN..VIVT HQLYNVLIT QLYN.AIIT
Neu
Neu Pd PS3
Yeast Beef Human Mouse Dros PIs Xen Den Neu Pd PS3
•
.
.
.
•
•
.
.
GG
51
52
57
UPUMDQA'T
89
P
P
89
P P
90 90 93
ur-J�"""'P P
96
\llPI.lMI�P vWr.lMtljAP \llPI.lMI�P
90 90
90
LWN.. VVVT LYNE .._ V�L�T������..�______
112 ���.R 100
L.....VESGAG M.....VEAGAG M.....VEAGAG M.....VEAGAG M ••... VENGAG G..... VESGAG G..... VEAGAS L.....VEVGSG
C
. . . . . IEGGAG LSPGGSDQPGAG
FLGGAPD . . . . .
124
125 125 125 124 125 125 128 131 152 135
CYTOCHROME
Yeast Beef Human Mouse Dros PIs Xen Den Neu
Annu. Rev. Biochem. 1990.59:569-596. Downloaded from www.annualreviews.org Access provided by University of Pittsburgh on 02/11/15. For personal use only.
Pd PS3
Yeast Beef Human Mouse Dros PIs Xen Den Neu Pd PS3
Yeast
Beef
Human Mouse Dros PIs Xen
IYP LYP SYA
GNLAHAGAS GITSHSGGA GVQSHSGPS TTEA .. GYA LDSKAHHG.
��� �� 'lj ��lt�t
575
LAlFALHLTSISSLLGA LTIFSLHLAGVSSILGA LTI F SL H LAGV S S I LGA LTlFSLHLAGVSSILGA LAIFSLHLAGISSILGA LAIFSLHLAGASSlLAS TIFSLHLAGASSILAS AISSLHLSGVSSILGS AIFALHLSGVSSLLGS AIFAVHVSGATSLLGAI YTLGL lSGFGTIMGAI
I 164 I 165
I 165
I 165 I 164 I
165
I 165
I 168
I 171
190 174
204 205 205 205
TNGMTMHKLP PPAMSQYQTP PPAMT QYQTP PPAMTQYQTP STGISLDRMP
Gil.
204
Gil. 205
TPGMSFDRLP
PPAMSQYQTP GLGMTMHRSP
AG 205 208 TPGIRLHKLA 211 ��Tl� M�APGMTLFKIP 230 ���r,APGMTFMRMP�� ���__����__� 214 ITMLL
PILYQ PILYQ PILYQ PILYQH •. "'IN..·..· PILFQ PVLYQHO."W..'"· P I LYQH •. "WI
