Quarterly Reviews of Biophysics 25, 3 (1992), pp. 253-324 Printed in Great Britain
253
The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains JOHN E. WALKER MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
1.
INTRODUCTION
254
1.1 Distribution of complex I 255 1.2 Nomenclature of subunits of bovine complex I encoded in nuclear DNA 257 2.
PURIFICATION AND RESOLUTION OF COMPLEX I
258
2.1 Purification 258 2.2 Resolution of complex I with chaotropes and detergents 260 2.3 Sequence analysis of subunits of complex I 262 2.3.1 Subunits synthesized in the mitochondrion 262 2.3.2 Subunits encoded in the nucleus 263 2.4 Molecular weights and post-translational modifications of subunits of bovine complex I 263 2.5 Molecular weight of complex I 265 3. STRUCTURAL FEATURES OF COMPLEX I 266
3.1 Structural relationship to a bacterial NAD+-reducing hydrogenase 266 3.2 NADH and FMN binding sites 267 3.3 Iron—sulphur proteins 271 3.3.1 Tetranuclear clusters 274 3.3.2 Binuclear clusters 276 3.3.3 Other possible iron-sulphur proteins 276 3.4 Quinone binding sites 277 3.5 Subunits involved in proton translocation 278 3.6 The extrinsic membrane domain of complex I 279 3.7 The intrinsic membrane domain 282 3.8 Structural models of complex I 285 4.
MECHANISM OF COMPLEX I
285
4.1 The coupling ratio 285 4.2 The electron pathway and coupling of electron transfer to proton export 285 5. IS THERE A COMPLEX I IN CHLORO PL ASTS ? 2 8 9
5.1 Chlororespiration 289 5.2 Homologues of complex I genes in chloroplast genomes 291 6. EVOLUTION OF COMPLEX I 292 12
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John E. Walker 6.1 Modular evolution of multi-subunit assemblies 292 6.2 Loci containing complex I genes in bacteria and chloroplasts 295 6.2.1 Bacterial NDH-1 genes 295 6.2.2 Subunits related to complex I in formate hydrogen lyase 295 6.2.3 Significance of gene orders 295 7.
SUBUNIT COMPLEXITY OF MITOCHONDRIAL COMPLEX I
296
7.1 Presence of an acyl carrier protein in mitochondrial complex I 297 7.2 Other proposed roles of complex I subunits 299 8. MEDICAL ASPECTS OF COMPLEX I
8.1 8.2 8.3 8.4
9. ACKNOWLEDGEMENTS IO.
I.
299
Diseases associated with mitochondrial DNA mutations 299 Involvement of mitochondria in ageing and cell death 302 Parkinson's disease 302 Huntington's disease 304
REFERENCES
304
305
INTRODUCTION
The inner membranes of mitochondria contain three multi-subunit enzyme complexes that act successively to transfer electrons from NADH to oxygen, which is reduced to water (Fig. i). The first enzyme in the electron transfer chain, NADH:ubiquinone oxidoreductase (or complex I), is the subject of this review. It removes electrons from NADH and passes them via a series of enzyme-bound redox centres (FMN and Fe-S clusters) to the electron acceptor ubiquinone. For each pair of electrons transferred from NADH to ubiquinone it is usually considered that four protons are removed from the matrix (see section 4.1 for further discussion of this point). The review is concerned mainly with the structure, mechanism and evolution of complex I, and is based upon recent information from three main sources. Firstly, the primary structures of nearly all of the 40 or more different subunits that make up the bovine mitochondrial enzyme have been determined, and sequences are known for some of the subunits of the enzyme from Neurospora crassa mitochondria, and from bacterial sources. The sequences provide important clues about the locations of prosthetic groups, and have revealed hitherto unsuspected relationships with other enzymes, which contribute to an understanding of the functions and the evolution of parts of complex I. They also suggest associations between some of the central subunits of the enzyme. Secondly, bovine complex I has been resolved into defined sub-complexes with detergents, and a smaller form of the JV. crassa enzyme has been produced by inhibition of mitochondrial protein synthesis. Thirdly, electron microscopic studies of the N. crassa enzyme have provided a low resolution outline of the enzyme's structure. Taken together with past work on the characterization of prosthetic groups in particular, these advances in knowledge of complex I provide the beginnings of a structural framework in which the mechanism of the enzyme
The NADH:ubiquinone oxidoreductase of respiratory chains
255
4H +
H+ + NADH
Cytoplasm 4H + Complex I
cyt Ac,
cyt aa3
Fig. 1. Electron transfer coupled to proton translocation in the mitochondrial electron transfer chain. The redox energy difference (1-2 V at neutral pH) is converted into an electrochemical proton potential gradient A/tH+ by translocation of protons and electron equivalents across the membrane. A/*H+ is harnessed by ATP synthase, another multisubunit enzyme in the inner membrane, to drive the synthesis of ATP from ADP and inorganic phosphate. n[Fe-S] denotes an undefined number of iron-sulphur clusters (but at least 4) in complex I. The coupling mechanism in complex I is not known, but possible mechanisms are discussed in the text (Section 4). For discussions of the mechanisms of cytochrome 6ct (complex III) and cytochrome aa3 (complex IV) see Trumpower (1990) and Babcock & Wikstrom (1992), respectively.
can be considered. They also have medical implications since defects in complex I are associated with a number of human diseases. Other reviews about complex I have emphasized different aspects such as its iron-sulphur clusters (Beinert & Albracht, 1982), sites of action of inhibitors (Singer & Ramsay, 1992), and mechanistic models (Ragan, 1990; Weiss & Friedrich, 1991). Advances with the N. crassa enzyme have been described by Weiss et al. (1991). A general review by Ragan (1987) provides a valuable general background to the field, but in structural aspects it has been largely superseded by more recent accounts. i. i Distribution of complex I In common with other respiratory enzymes, a minority of the subunits of mammalian complex I are the products of mitochondrial genes and are synthesized in the organelle (see Table i). These are known as subunits NDi—ND6 and subunit ND4L, and all seven of them are hydrophobic intrinsic membrane proteins. Mitochondrial DNA sequences have been determined in a wide range of eukaryotes including mammals (Anderson et al. 1981, 1982; Bibb etal. 1981; Gadaleta et al. 1989; Arnasonet al. 1991), birds (Desjardins & Morais, 12-2
256
John E. Walker
Table 1. Numbers of subunits and locations of genes of respiratory complexes from bovine heart mitochondria and complexities of bacterial counterparts Mitochondria Complex
Nucleus
mt-DNA (subunit names) Bacteria*
References^
complex I cytochrome bcy cytochrome oxidase ATP synthase
>34
7(NDi-ND6, ND4L) 1 (cytochrome b) 3 (COI-COIII) 2 (ATPase-6, A6L)
1, 2
10 10 12
> IO a
2"-" or 40 3-7 3" 8 8* or o" 9, 10
* Exemplified by: "Paracoccus denitrificans; bRhodospirillum rubrum; cRhodobacter sphaeroides; dE. coli.
f References: (1) Walker et al. (1992); (2) Yagi (1991); (3) Engel et al. (1983); (4) Yang & Trumpower (1986); (5) Purvis et al. (1990); (6) Andrews et al. (1990); (7) Kriauciunas (1989); (8) Saraste (1990); (9) Walker et al. (1991); (10) Falk & Walker (1988).
1990), fish (Johansen et al. 1990), insects (Clary & Wolstenholme, 1985; Garesse, 1988), amphibians (Roe et al. 1985), invertebrates including sea urchins (Jacobs et al. 1988; Cantatore et al. 1989), nematode worms (Okimoto et al. 1992) and brine shrimps (Batuecas et al. 1988), protozoans (Pritchard et al. 1990), algae (Denovan-Wright & Lee, 1992), fungi (Burger & Werner, 1986; Nelson & Macino, 1987; Cummings et al. 1990), and higher plants (Stern et al. 1986; Wintz et al. 1989; Suzuki et al. 1991; Oda et al. 1992). These examples, and others that are not mentioned here, contain in their mitochondrial genomes homologues of some or all of the mitochondrial genes ND1-ND6 and ND4L, and demonstrate the presence of complex I in a wide variety of species. The yeast Saccharomyces cerevisiae has no ND genes in its mitochondrial DNA and is a notable exception. The significance of genes related to the mitochondrial ND genes in the chloroplast genomes of higher plants will be dealt with later (section 5). Complex I has been purified from bovine and N. crassa mitochondria, and these are by far the most extensively studied examples of the enzyme. Biochemical studies have also been conducted on complex I from a variety of mammals, from blow-fly, plants, and the yeast, Candida utilis (see Ragan, 1987). Two classes of NADH-ubiquinone oxidoreductases, known as NDH-i and NDH-2, are found in bacteria (Yagi, 1991). The NDH-i class are energy conserving enzymes. They have been detected in Paracoccus denitrificans (Yagi, 1986, 1991), Thermus thermophilus (Yagi et al. 1988), E. coli (Hayashi et al. 1989), Rhodobacter capsulatus (Baccarini-Melandri et al. 1973) and Synechocystis sp. PCC 6803 (Berger et al. 1991). It has been suggested that the NDH-i enzymes from P. denitrificans and T. thermophilus are the bacterial counterparts of mitocondrial complex I. Like the mitochondrial enzymes they contain noncovalently bound FMN and Fe-S clusters as prosthetic groups, they pump protons, and their activity can be inhibited by rotenone, a classical inhibitor of NADH-ubiquinone reduction in mitochondria. However, neither enzyme has
The NADH:ubiquinone oxidoreductase of respiratory chains
257
been purified in a rotenone sensitive state, reconstituted and demonstrated to translocate protons. Therefore, the subunit complexities of the bacterial enzymes are not known. An NADH:ubiquinone oxidoreductase activity has been purified from P. denitrificans by extraction of membranes with the chaotropic agent, sodium bromide (Yagi, 1986). This preparation contains at least 10 polypeptides, but it has not been shown to translocate protons and, although it clearly has features in common with mitochondrial complex I, it is quite likely to represent a fragment of the bacterial complex rather than being the complete entity. A proton-translocating NADH:ubiquinone oxidoreductase, in which at least 10 polypeptides can be discerned, has been isolated from T. thermophilus (Yagi et al. 1988), but the preparation is rotenone insensitive and again may not represent the entire enzyme complex (see Berks & Ferguson, 1991, for a fuller discussion of the complexity and of the problems of purifying bacterial NDH-i enzymes). A sodium-pumping NADH-quinone oxidoreductase, NQRi, is found in marine organisms and enteric bacteria (Unemoto & Hayashi, 1989; Dimroth & Thomer, 1989). The NQRi from Vibrio alginolyticus consists of three subunits a, /? and y. The a and (5 subunits are both flavoproteins and contain FMN and FAD, respectively (Hayashi & Unemoto, 1986, 1987). The NDH-2 class appears to be unrelated to complex I, and is mentioned only briefly here. Its members have a single polypeptide chain and contain noncovalently bound FAD, but no Fe-S clusters; they do not pump protons and are unaffected by inhibitors of complex I, such as rotenone and piericidin (de Vries & Marres, 1987). Both NDH-i and NDH-2 are present in T. thermophilus (Yagi et al. 1988), and E. coli (Hayashi et al. 1989). An NDH-2, but not an NDH-i enzyme, is found in the mitochondria of the yeast, 5. cerevisiae (de Vries & Grivell, 1988). NAD(P)H:quinone oxidoreductases (NQO), formerly known as DTdiaphorases, are flavoproteins found mainly in the cytoplasmic fraction of liver. Their physiological function is not understood, but they could be involved in the detoxification of non-physiological quinones. They are single subunit water soluble enzymes and are not evidently related to any component of complex I (Jaiswal et al. 1990). 1.2 Nomenclature of subunits of bovine complex I encoded in nuclear DNA Subunits belonging to the flavoprotein (FP) fraction of complex I (see section 2.2 below), are referred to as 51 kDa (FP), 24 kDa (FP) and iokDa (FP), and six proteins found in the iron-sulphur protein (IP) fraction of complex I (see below; Galante & Hatefi, 1979; Ragan, 1987), are called 75 kDa (IP), 49 kDa (IP), 30 kDa (IP), 18 kDa (IP), 15 kDa (IP) and 13 kDa (IP). Two other constituents of complex I are known as the 39 and 42 kDa subunits. Otherwise, proteins with unmodified a-amino groups are named according to the one letter code of the sequence of amino acids 1—4. Nine further nuclear encoded proteins with modified a-amino groups and estimated molecular weights of 8, 9, 12, 13, 14, 15, 17, 18 and 22 kDa are called B8, B9, B12, B13, B14, B15, B17, B18 and B22, respectively. The nomenclature for nuclear coded subunits is considered to be an interim one
258
John E. Walker 0)
15—-*
51 — 7, and so the sequences that are aligned in Fig. 5 are most likely to be those that are involved in forming the ADP-binding pocket in the NAD + binding sites in these proteins. In the two sites of deviation from the fingerprint in the bovine 51 kDa subunit (amino acids 76 and 79) small hydrophobic amino acids are expected, but glycine and tryptophan, respectively are found. The same substitutions are found in the
The NADH:ubiquinone oxidoreductase of respiratory chains Protein
Amino a c i d s e q u e n c e
Structure UM (21-521 ADH (193-223) G3PDH L (1-31) Consensus B. t a u r u s 5 1 (61-99) N. crassa 50 (63-104) P. denitrificans 50 (48-86) Hox F (213-250)
PA cB ~ |B N K I T Y V G - V G . A V G M A C A I S I L M K D L A D E Y A L V D S I C A I F 5 - LfiGVJ L S V I B G C K A A G A A R I I G V D S i S I G I N G - F S R I G R L V i R A f i L S C G i Q V J A I K D A * * . . . * * * * A K J S . G L R G - R G G A G F P T G L K W S F M N K P S D G R P K Y L V Y N A D E K A S G L R G . - R S G A G . F P S G L K W S F M N F K D W D K D D K P R Y L V Y N A D E K A S G L R S - R G G A G F P T G M K W S F M P K E S D G R P S Y L V I N A D E V D S R L R G - R G G A G F S T G L K W R L C R D A E S E Q K Y V I C N A D E
B. N. H. B.
I V A H I A W £ £ « S £
t a u r u s 3 9 (19-49) crassa 40 (26-56) sapiens 3 b h s d (3-35) t a u r u s 3 t h s d (3-35)
Yeast ADH (171-202)
T Y T S L y L J
F G F G T G T C
A T G F L G R Y V y N H L G R M G A T f i Q L C H J I V K B L G R Q G A G G F L G Q R I J R L L V K E K C G S P L G 0 B I I C L 1 V E E K
HWYAISGAAGGLGSLAYQYAKAMG
269 Score
S C E L K D L Q
11 10 11 9 8 8 8
Q I I J P H T I V I P F E J R Y L B E I R Y L D
9 10 11 11
YRYLGID
9
Fig. 5. Sequences in dehydrogenases known to bind the ADP portion of NADH, compared with possible ADP-binding regions in the 51 and 39 kDa subunits of complex I and in related proteins. The ADP-binding region from three hydrogenases of known structure are shown above. These are dogfish lactate dehydrogenase (LDH; White et al. 1976; Taylor, 1977), horse liver alcohol dehydrogenase (ADH; Eklund et al. 1981), and lobster glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Moras et al. 1975). The secondary structure of the fold is indicated above these sequences. Beneath them is a consensus fingerprint for the site (Wierenga et al. 1986) in which the following symbols are used: A, basic or hydrophilic residue; -j^-, small and hydrophobic amino acid (A, I, L, V, M, C); # , invariant glycine; A , acidic residue (D, E). The score indicates the number of fingerprint positions at which the correct type of residue is found. Residues in agreement with the fingerprint are underlined. Under the consensus are regions of the 51 and 50 kDa subunits of bovine and AT. crassa complex I, respectively (Pilkington et al. 1991a; Preis et al. 1991), of the 50 kDa subunit of P. denitrificans NADH dehydrogenase (Xu et al. 1991a), and of the A. eutrophus HoxF protein (Tran-Betcke et al. 1990) that may form the NADH-binding sites in these enzymes. Also shown are regions of the 39 and 40 kDa subunits of bovine (Fearnley et al. 1991) and N. crassa (Rohlen et al. 1991) complex I and of the human and bovine 3/?-hydroxy-5-ene steroid dehydrogenases (Luu-The et al. 1989; Zhao et al. 1989) that may also contain NADH-binding sites. The alignment with the fingerprint requires the insertion of an amino acid at residue 8. This extra amino acid is present in a turn in the region that forms the NADH-binding site in yeast alcohol dehydrogenase (Jornvall, 1977).
NADH-binding sites of dihydrolipoamide dehydrogenase in several species (Scrutton et al. 1990). In NADH-binding proteins of known structure, the nicotinamide-ribose moiety is also bound by a /?-a-/? fold similar to the ADP-binding fold, but the sequences that form them are not evidently conserved, and their structures are more variable than those of ADP-binding sites (Rossman et al. 1975). Therefore, the parts of the bovine 5 1 kDa subunit (and its counterparts in other species) that fold into the nicotinamide-ribose pocket cannot be predicted from the sequence. However, it is likely from consideration of known structures that these sequences lie to the C-terminal side of those that are proposed to form the ADP pockets. FMN is usually considered to be the most likely immediate oxidant of NADH. Its standard reduction potential is intermediate between those of NADH and the most electronegative of the EPR visible iron-sulphur clusters, and it can take up two electrons at a time and release them one at a time to a one electron acceptor such as an Fe-S cluster. Since NADH binds to the 51 kDa subunit it seems likely
270
John E. Walker
that FMN will be bound to the same polypeptide, the only other possibilities being the 24 kDa (FP) and 10 kDa (FP) subunits. There is no direct evidence to support this suggestion, and little or nothing is known about the location of its binding site. Three different types of FMN-binding site have been described in flavoenzymes of known structure (Mathews, 1991). One class is the (/?
253
The NADH:ubiquinone oxidoreductase (complex I) of respiratory chains JOHN E. WALKER MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
1.
INTRODUCTION
254
1.1 Distribution of complex I 255 1.2 Nomenclature of subunits of bovine complex I encoded in nuclear DNA 257 2.
PURIFICATION AND RESOLUTION OF COMPLEX I
258
2.1 Purification 258 2.2 Resolution of complex I with chaotropes and detergents 260 2.3 Sequence analysis of subunits of complex I 262 2.3.1 Subunits synthesized in the mitochondrion 262 2.3.2 Subunits encoded in the nucleus 263 2.4 Molecular weights and post-translational modifications of subunits of bovine complex I 263 2.5 Molecular weight of complex I 265 3. STRUCTURAL FEATURES OF COMPLEX I 266
3.1 Structural relationship to a bacterial NAD+-reducing hydrogenase 266 3.2 NADH and FMN binding sites 267 3.3 Iron—sulphur proteins 271 3.3.1 Tetranuclear clusters 274 3.3.2 Binuclear clusters 276 3.3.3 Other possible iron-sulphur proteins 276 3.4 Quinone binding sites 277 3.5 Subunits involved in proton translocation 278 3.6 The extrinsic membrane domain of complex I 279 3.7 The intrinsic membrane domain 282 3.8 Structural models of complex I 285 4.
MECHANISM OF COMPLEX I
285
4.1 The coupling ratio 285 4.2 The electron pathway and coupling of electron transfer to proton export 285 5. IS THERE A COMPLEX I IN CHLORO PL ASTS ? 2 8 9
5.1 Chlororespiration 289 5.2 Homologues of complex I genes in chloroplast genomes 291 6. EVOLUTION OF COMPLEX I 292 12
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John E. Walker 6.1 Modular evolution of multi-subunit assemblies 292 6.2 Loci containing complex I genes in bacteria and chloroplasts 295 6.2.1 Bacterial NDH-1 genes 295 6.2.2 Subunits related to complex I in formate hydrogen lyase 295 6.2.3 Significance of gene orders 295 7.
SUBUNIT COMPLEXITY OF MITOCHONDRIAL COMPLEX I
296
7.1 Presence of an acyl carrier protein in mitochondrial complex I 297 7.2 Other proposed roles of complex I subunits 299 8. MEDICAL ASPECTS OF COMPLEX I
8.1 8.2 8.3 8.4
9. ACKNOWLEDGEMENTS IO.
I.
299
Diseases associated with mitochondrial DNA mutations 299 Involvement of mitochondria in ageing and cell death 302 Parkinson's disease 302 Huntington's disease 304
REFERENCES
304
305
INTRODUCTION
The inner membranes of mitochondria contain three multi-subunit enzyme complexes that act successively to transfer electrons from NADH to oxygen, which is reduced to water (Fig. i). The first enzyme in the electron transfer chain, NADH:ubiquinone oxidoreductase (or complex I), is the subject of this review. It removes electrons from NADH and passes them via a series of enzyme-bound redox centres (FMN and Fe-S clusters) to the electron acceptor ubiquinone. For each pair of electrons transferred from NADH to ubiquinone it is usually considered that four protons are removed from the matrix (see section 4.1 for further discussion of this point). The review is concerned mainly with the structure, mechanism and evolution of complex I, and is based upon recent information from three main sources. Firstly, the primary structures of nearly all of the 40 or more different subunits that make up the bovine mitochondrial enzyme have been determined, and sequences are known for some of the subunits of the enzyme from Neurospora crassa mitochondria, and from bacterial sources. The sequences provide important clues about the locations of prosthetic groups, and have revealed hitherto unsuspected relationships with other enzymes, which contribute to an understanding of the functions and the evolution of parts of complex I. They also suggest associations between some of the central subunits of the enzyme. Secondly, bovine complex I has been resolved into defined sub-complexes with detergents, and a smaller form of the JV. crassa enzyme has been produced by inhibition of mitochondrial protein synthesis. Thirdly, electron microscopic studies of the N. crassa enzyme have provided a low resolution outline of the enzyme's structure. Taken together with past work on the characterization of prosthetic groups in particular, these advances in knowledge of complex I provide the beginnings of a structural framework in which the mechanism of the enzyme
The NADH:ubiquinone oxidoreductase of respiratory chains
255
4H +
H+ + NADH
Cytoplasm 4H + Complex I
cyt Ac,
cyt aa3
Fig. 1. Electron transfer coupled to proton translocation in the mitochondrial electron transfer chain. The redox energy difference (1-2 V at neutral pH) is converted into an electrochemical proton potential gradient A/tH+ by translocation of protons and electron equivalents across the membrane. A/*H+ is harnessed by ATP synthase, another multisubunit enzyme in the inner membrane, to drive the synthesis of ATP from ADP and inorganic phosphate. n[Fe-S] denotes an undefined number of iron-sulphur clusters (but at least 4) in complex I. The coupling mechanism in complex I is not known, but possible mechanisms are discussed in the text (Section 4). For discussions of the mechanisms of cytochrome 6ct (complex III) and cytochrome aa3 (complex IV) see Trumpower (1990) and Babcock & Wikstrom (1992), respectively.
can be considered. They also have medical implications since defects in complex I are associated with a number of human diseases. Other reviews about complex I have emphasized different aspects such as its iron-sulphur clusters (Beinert & Albracht, 1982), sites of action of inhibitors (Singer & Ramsay, 1992), and mechanistic models (Ragan, 1990; Weiss & Friedrich, 1991). Advances with the N. crassa enzyme have been described by Weiss et al. (1991). A general review by Ragan (1987) provides a valuable general background to the field, but in structural aspects it has been largely superseded by more recent accounts. i. i Distribution of complex I In common with other respiratory enzymes, a minority of the subunits of mammalian complex I are the products of mitochondrial genes and are synthesized in the organelle (see Table i). These are known as subunits NDi—ND6 and subunit ND4L, and all seven of them are hydrophobic intrinsic membrane proteins. Mitochondrial DNA sequences have been determined in a wide range of eukaryotes including mammals (Anderson et al. 1981, 1982; Bibb etal. 1981; Gadaleta et al. 1989; Arnasonet al. 1991), birds (Desjardins & Morais, 12-2
256
John E. Walker
Table 1. Numbers of subunits and locations of genes of respiratory complexes from bovine heart mitochondria and complexities of bacterial counterparts Mitochondria Complex
Nucleus
mt-DNA (subunit names) Bacteria*
References^
complex I cytochrome bcy cytochrome oxidase ATP synthase
>34
7(NDi-ND6, ND4L) 1 (cytochrome b) 3 (COI-COIII) 2 (ATPase-6, A6L)
1, 2
10 10 12
> IO a
2"-" or 40 3-7 3" 8 8* or o" 9, 10
* Exemplified by: "Paracoccus denitrificans; bRhodospirillum rubrum; cRhodobacter sphaeroides; dE. coli.
f References: (1) Walker et al. (1992); (2) Yagi (1991); (3) Engel et al. (1983); (4) Yang & Trumpower (1986); (5) Purvis et al. (1990); (6) Andrews et al. (1990); (7) Kriauciunas (1989); (8) Saraste (1990); (9) Walker et al. (1991); (10) Falk & Walker (1988).
1990), fish (Johansen et al. 1990), insects (Clary & Wolstenholme, 1985; Garesse, 1988), amphibians (Roe et al. 1985), invertebrates including sea urchins (Jacobs et al. 1988; Cantatore et al. 1989), nematode worms (Okimoto et al. 1992) and brine shrimps (Batuecas et al. 1988), protozoans (Pritchard et al. 1990), algae (Denovan-Wright & Lee, 1992), fungi (Burger & Werner, 1986; Nelson & Macino, 1987; Cummings et al. 1990), and higher plants (Stern et al. 1986; Wintz et al. 1989; Suzuki et al. 1991; Oda et al. 1992). These examples, and others that are not mentioned here, contain in their mitochondrial genomes homologues of some or all of the mitochondrial genes ND1-ND6 and ND4L, and demonstrate the presence of complex I in a wide variety of species. The yeast Saccharomyces cerevisiae has no ND genes in its mitochondrial DNA and is a notable exception. The significance of genes related to the mitochondrial ND genes in the chloroplast genomes of higher plants will be dealt with later (section 5). Complex I has been purified from bovine and N. crassa mitochondria, and these are by far the most extensively studied examples of the enzyme. Biochemical studies have also been conducted on complex I from a variety of mammals, from blow-fly, plants, and the yeast, Candida utilis (see Ragan, 1987). Two classes of NADH-ubiquinone oxidoreductases, known as NDH-i and NDH-2, are found in bacteria (Yagi, 1991). The NDH-i class are energy conserving enzymes. They have been detected in Paracoccus denitrificans (Yagi, 1986, 1991), Thermus thermophilus (Yagi et al. 1988), E. coli (Hayashi et al. 1989), Rhodobacter capsulatus (Baccarini-Melandri et al. 1973) and Synechocystis sp. PCC 6803 (Berger et al. 1991). It has been suggested that the NDH-i enzymes from P. denitrificans and T. thermophilus are the bacterial counterparts of mitocondrial complex I. Like the mitochondrial enzymes they contain noncovalently bound FMN and Fe-S clusters as prosthetic groups, they pump protons, and their activity can be inhibited by rotenone, a classical inhibitor of NADH-ubiquinone reduction in mitochondria. However, neither enzyme has
The NADH:ubiquinone oxidoreductase of respiratory chains
257
been purified in a rotenone sensitive state, reconstituted and demonstrated to translocate protons. Therefore, the subunit complexities of the bacterial enzymes are not known. An NADH:ubiquinone oxidoreductase activity has been purified from P. denitrificans by extraction of membranes with the chaotropic agent, sodium bromide (Yagi, 1986). This preparation contains at least 10 polypeptides, but it has not been shown to translocate protons and, although it clearly has features in common with mitochondrial complex I, it is quite likely to represent a fragment of the bacterial complex rather than being the complete entity. A proton-translocating NADH:ubiquinone oxidoreductase, in which at least 10 polypeptides can be discerned, has been isolated from T. thermophilus (Yagi et al. 1988), but the preparation is rotenone insensitive and again may not represent the entire enzyme complex (see Berks & Ferguson, 1991, for a fuller discussion of the complexity and of the problems of purifying bacterial NDH-i enzymes). A sodium-pumping NADH-quinone oxidoreductase, NQRi, is found in marine organisms and enteric bacteria (Unemoto & Hayashi, 1989; Dimroth & Thomer, 1989). The NQRi from Vibrio alginolyticus consists of three subunits a, /? and y. The a and (5 subunits are both flavoproteins and contain FMN and FAD, respectively (Hayashi & Unemoto, 1986, 1987). The NDH-2 class appears to be unrelated to complex I, and is mentioned only briefly here. Its members have a single polypeptide chain and contain noncovalently bound FAD, but no Fe-S clusters; they do not pump protons and are unaffected by inhibitors of complex I, such as rotenone and piericidin (de Vries & Marres, 1987). Both NDH-i and NDH-2 are present in T. thermophilus (Yagi et al. 1988), and E. coli (Hayashi et al. 1989). An NDH-2, but not an NDH-i enzyme, is found in the mitochondria of the yeast, 5. cerevisiae (de Vries & Grivell, 1988). NAD(P)H:quinone oxidoreductases (NQO), formerly known as DTdiaphorases, are flavoproteins found mainly in the cytoplasmic fraction of liver. Their physiological function is not understood, but they could be involved in the detoxification of non-physiological quinones. They are single subunit water soluble enzymes and are not evidently related to any component of complex I (Jaiswal et al. 1990). 1.2 Nomenclature of subunits of bovine complex I encoded in nuclear DNA Subunits belonging to the flavoprotein (FP) fraction of complex I (see section 2.2 below), are referred to as 51 kDa (FP), 24 kDa (FP) and iokDa (FP), and six proteins found in the iron-sulphur protein (IP) fraction of complex I (see below; Galante & Hatefi, 1979; Ragan, 1987), are called 75 kDa (IP), 49 kDa (IP), 30 kDa (IP), 18 kDa (IP), 15 kDa (IP) and 13 kDa (IP). Two other constituents of complex I are known as the 39 and 42 kDa subunits. Otherwise, proteins with unmodified a-amino groups are named according to the one letter code of the sequence of amino acids 1—4. Nine further nuclear encoded proteins with modified a-amino groups and estimated molecular weights of 8, 9, 12, 13, 14, 15, 17, 18 and 22 kDa are called B8, B9, B12, B13, B14, B15, B17, B18 and B22, respectively. The nomenclature for nuclear coded subunits is considered to be an interim one
258
John E. Walker 0)
15—-*
51 — 7, and so the sequences that are aligned in Fig. 5 are most likely to be those that are involved in forming the ADP-binding pocket in the NAD + binding sites in these proteins. In the two sites of deviation from the fingerprint in the bovine 51 kDa subunit (amino acids 76 and 79) small hydrophobic amino acids are expected, but glycine and tryptophan, respectively are found. The same substitutions are found in the
The NADH:ubiquinone oxidoreductase of respiratory chains Protein
Amino a c i d s e q u e n c e
Structure UM (21-521 ADH (193-223) G3PDH L (1-31) Consensus B. t a u r u s 5 1 (61-99) N. crassa 50 (63-104) P. denitrificans 50 (48-86) Hox F (213-250)
PA cB ~ |B N K I T Y V G - V G . A V G M A C A I S I L M K D L A D E Y A L V D S I C A I F 5 - LfiGVJ L S V I B G C K A A G A A R I I G V D S i S I G I N G - F S R I G R L V i R A f i L S C G i Q V J A I K D A * * . . . * * * * A K J S . G L R G - R G G A G F P T G L K W S F M N K P S D G R P K Y L V Y N A D E K A S G L R G . - R S G A G . F P S G L K W S F M N F K D W D K D D K P R Y L V Y N A D E K A S G L R S - R G G A G F P T G M K W S F M P K E S D G R P S Y L V I N A D E V D S R L R G - R G G A G F S T G L K W R L C R D A E S E Q K Y V I C N A D E
B. N. H. B.
I V A H I A W £ £ « S £
t a u r u s 3 9 (19-49) crassa 40 (26-56) sapiens 3 b h s d (3-35) t a u r u s 3 t h s d (3-35)
Yeast ADH (171-202)
T Y T S L y L J
F G F G T G T C
A T G F L G R Y V y N H L G R M G A T f i Q L C H J I V K B L G R Q G A G G F L G Q R I J R L L V K E K C G S P L G 0 B I I C L 1 V E E K
HWYAISGAAGGLGSLAYQYAKAMG
269 Score
S C E L K D L Q
11 10 11 9 8 8 8
Q I I J P H T I V I P F E J R Y L B E I R Y L D
9 10 11 11
YRYLGID
9
Fig. 5. Sequences in dehydrogenases known to bind the ADP portion of NADH, compared with possible ADP-binding regions in the 51 and 39 kDa subunits of complex I and in related proteins. The ADP-binding region from three hydrogenases of known structure are shown above. These are dogfish lactate dehydrogenase (LDH; White et al. 1976; Taylor, 1977), horse liver alcohol dehydrogenase (ADH; Eklund et al. 1981), and lobster glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Moras et al. 1975). The secondary structure of the fold is indicated above these sequences. Beneath them is a consensus fingerprint for the site (Wierenga et al. 1986) in which the following symbols are used: A, basic or hydrophilic residue; -j^-, small and hydrophobic amino acid (A, I, L, V, M, C); # , invariant glycine; A , acidic residue (D, E). The score indicates the number of fingerprint positions at which the correct type of residue is found. Residues in agreement with the fingerprint are underlined. Under the consensus are regions of the 51 and 50 kDa subunits of bovine and AT. crassa complex I, respectively (Pilkington et al. 1991a; Preis et al. 1991), of the 50 kDa subunit of P. denitrificans NADH dehydrogenase (Xu et al. 1991a), and of the A. eutrophus HoxF protein (Tran-Betcke et al. 1990) that may form the NADH-binding sites in these enzymes. Also shown are regions of the 39 and 40 kDa subunits of bovine (Fearnley et al. 1991) and N. crassa (Rohlen et al. 1991) complex I and of the human and bovine 3/?-hydroxy-5-ene steroid dehydrogenases (Luu-The et al. 1989; Zhao et al. 1989) that may also contain NADH-binding sites. The alignment with the fingerprint requires the insertion of an amino acid at residue 8. This extra amino acid is present in a turn in the region that forms the NADH-binding site in yeast alcohol dehydrogenase (Jornvall, 1977).
NADH-binding sites of dihydrolipoamide dehydrogenase in several species (Scrutton et al. 1990). In NADH-binding proteins of known structure, the nicotinamide-ribose moiety is also bound by a /?-a-/? fold similar to the ADP-binding fold, but the sequences that form them are not evidently conserved, and their structures are more variable than those of ADP-binding sites (Rossman et al. 1975). Therefore, the parts of the bovine 5 1 kDa subunit (and its counterparts in other species) that fold into the nicotinamide-ribose pocket cannot be predicted from the sequence. However, it is likely from consideration of known structures that these sequences lie to the C-terminal side of those that are proposed to form the ADP pockets. FMN is usually considered to be the most likely immediate oxidant of NADH. Its standard reduction potential is intermediate between those of NADH and the most electronegative of the EPR visible iron-sulphur clusters, and it can take up two electrons at a time and release them one at a time to a one electron acceptor such as an Fe-S cluster. Since NADH binds to the 51 kDa subunit it seems likely
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John E. Walker
that FMN will be bound to the same polypeptide, the only other possibilities being the 24 kDa (FP) and 10 kDa (FP) subunits. There is no direct evidence to support this suggestion, and little or nothing is known about the location of its binding site. Three different types of FMN-binding site have been described in flavoenzymes of known structure (Mathews, 1991). One class is the (/?
