Photosynthesis Research 33: 121-136, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands. Minireview

Cytochrome

and b6f complexes of photosynthetic membranes

Richard Malkin Department of Plant Biology, University of California, Berkeley, CA 94720, USA Received 1 September 1991; accepted in revised form 12 March 1992

Key words: cation

cytochrome f, cytochrome b, Rieske iron-sulfur protein, plastoquinone, proton translo-

Abstract

All photosynthetic membranes contain a cytochrome bc 1 or b6f complex that catalyzes the oxidation of quinols and the reduction of a high-potential electron carrier, such as cytochrome c 2 or plastocyanin. The cytochrome complex also functions in the translocation of protons across the membrane and as a consequence, establishes the proton motive force that is used for the synthesis of ATP. The structure and function of the cytochrome complexes are first reviewed in this chapter. Amino acid sequence information for almost all of the protein subunits of these complexes is now available, and these allow for a detailed consideration of functional domains in the protein subunits and for a further discussion of the evolution of the cytochrome complex in photosynthetic organisms.

1. Introduction

Photosynthetic membranes contain a number of integral membrane protein complexes that are involved in energy conversion reactions. Most notable are the chlorophyll-containing photochemical reaction center complexes that capture light energy and convert this energy into stabilized chemical products. In addition to these socalled photochemical complexes, all photosynthetic membranes also contain an electron transfer complex, known either as the cytochrome bc 1 or b6f complex, that converts the redox energy released during the oxidation of quinols into a gradient of protons across the membrane. This proton gradient is a high-energy source that serves as the driving force for the synthesis of ATP in these systems and because of this role, the cytochrome complex plays a critical role in cellular energy conversion (for recent reviews dealing with aspects of the structure and function of cytochrome bc] b 6f complexes, see Hauska et al. 1983, Gabellini 1988, Cramer et al. 1987, Trumpower 1990).

In terms of function and composition, the photosynthetic cytochrome complex is analogous to the extensively studied Complex III of the mitochondrial respiratory chain (Rieske 1976). This multi-subunit complex, localized in the inner mitochondrial membrane, had been characterized in the 1960s in a series of now classical biochemical studies that led to the isolation of an enzymatically active complex that functioned as a ubiquinol-cytochrome c oxidoreductase (de Vries and Marres 1987, Rich 1984, Trumpower and Katki 1979). It has only been in recent years that it has been realized that other energy-transducing membranes, such as those found in chloroplasts or bacteria, also contain a complex that is analogous in function and contains similar prosthetic groups involved in these functions (Hauska et al. 1983, Cramer et al. 1987, Malkin 1988). This chapter will consider the structure and function of the photosynthetic cytochrome complex and the identity of its key components that allow this functional role. A comparison of the cytochrome b¢ 1 complex from prokaryotic photo-

122 synthetic organisms with the cytochrome b6f complex from both prokaryotic and eukaryotic photosynthetic systems offers the possibility of considering evolutionary aspects of this electron transfer complex in biological systems.

2. Composition of the cytochrome complex In photosynthetic organisms the cytochrome complex functions by transfering electrons from quinol molecules, such as ubiquinol or plastoquinol, to high-potential protein electron acceptors, such as cytochrome c 2 or plastocyanin (Hurt and Hauska 1981). Accompanying this electron transfer sequence, protons are translocated across the membrane and in all cases, the ratio of protons translocated to electrons transferred (H+/e - ratio) is two (Hurt et al. 1983). Mechanistically, this is a complicated reaction since one 'extra' proton is translocated for every electron passed to the terminal electron acceptor. Since the reactions catalyzed by these complexes, either in prokaryotic or eukaryotic systems, are highly analogous, it is not surprising that the composition of these complexes are consistent with this view. Thus, cytochrome bc 1 and b6f complexes always contain a c-type high potential cytochrome (cytochrome c I in the bc 1 complex and cytochrome f in the b6f complex) and two b-type hemes that have different midpoint redox potentials (cytochrome b 6 in the b6f complex and cytochromes bh and bl in the bc 1 complex). One of the most unusual and distinguishing components in this complex is a high potential two iron-two sulfur protein, known as the Rieske iron-sulfur protein, after the inves-

tigator who was involved in the initial characterization of this electron transfer carrier in the mitochondrial complex (Rieske et al. 1964). This carrier is best identified on the basis of its low temperature EPR signal with a g-value of approximately 1.90. The unifying feature of all such electron transfer complexes is, therefore, the presence of four electron transfer groups, two of which have relatively high redox potentials (Em greater than +250mV) and two of which have relatively lower redox potentials (Em approximately 0 mV or lower). The latter two carriers are associated with the b-type hemes in the complex while the c-type cytochrome and the Rieske iron-sulfur protein are the high-potential carriers. These properties are summarized in Table 1. While the electron transfer component composition of the respective complexes is amazingly consistent, the protein subunits found in these complexes shows some interesting apparent differences. In the case of the cytochrome bc~ complex found in several bacteria, there are apparently only 3 protein subunits present, corresponding to the three electron carriers in the complex (Trumpower 1990, Malkin 1988). Because only a single subunit corresponds to the b-type cytochrome, the two hemes in this molecule must be bound to a single subunit. The b-cytochrome has a molecular mass of approximately 40 kDa, based on D N A sequencing of the gene for this subunit, while the c-type cytochrome has a molecular mass of approximately 30 kDa and the Rieske iron-sulfur subunit, a molecular mass of approximately 20kDa. An additional subunit has been reported in a corn-

Table 1. R e d o x components of cytochrome bc 1 and b 6 f complexes Electron carrier

E m value

Reference

c-type cytochrome bc 1 complex: cytochrome c 1 b 6f complex: cytochrome f

+ 270 mV + 340 mV

T'sai and Palmer (1983) Hurt and H a u s k a (1982a)

Rieske F e - S protein bc I complex b 6 f complex

+280 mV +290 mV

T'sai and Palmer (1983) Malkin (1986)

b-type cytochrome bc 1 complex: cytochrome b b 6 f complex: cytochrome b 6

heine heme heine heme

H L H L

= = = =

+50 mV - 60 mV - 5 0 mV - 170 mV

T'sai and Palmer (1983) T'sai and Palmer (1983) Baroli et al. (1991), H u r t and H a u s k a (1983) Baroli et al. (1991), H u r t and H a u s k a (1983)

123 synthetic cytochrome complexes and also contains similar data for the well-characterized mitochondrial Complex III. In the latter case, in addition to the prosthetic group binding subunits, there are approximately 6-8 additional subunits present in the complex that do not bind electron transfer components, and the function of these subunits is still undetermined. Complete amino acid sequences, derived from D N A sequences, are now available for all the subunits of these cytochrome complexes. These sequences have led to predictions of tertiary structures based on hydropathy profiles. It is clear that cytochrome b (or cytochrome b 6 plus subunit IV) is the most hydrophobic component of the complex and it has been predicted that this subunit contains at least 8 transmembrane spanning helices (Saraste 1984). The protein contains only four conserved histidine residues and these are believed to be ligands to the two heme groups in the molecule. The c-type cytochrome is considerably less hydrophobic and has been proposed to contain only a single transm e m b r a n e helix that crosses the membrane (Willey et al. 1984). The Rieske iron-sulfur protein is the least hydrophobic subunit of the complex and has been proposed to contain a single transmembrane helix although there is much less certainty concerning this assignment and recent results have suggested that in the case of Complex III, this subunit may be a more extrinsic subunit and not firmly imbedded in the lipid bilayer (Hartl et al. 1986, Gonzalez-

plex from a photosynthetic bacterium (Ljungdahl et al. 1987, Yu et al. 1984), but whether this subunit is an intrinsic component of this complex remains to be determined (see Yu and Yu 1991 for recent results on this subject). The cytochrome b 6 f complex from eukaryotic organisms as well as from cyanobacteria contains four protein subunits (Hauska et al. 1983, Malkin 1988). Again, the c-type cytochrome and the Riekse iron-sulfur subunit have molecular masses near those found in the complexes from photosynthetic bacteria (approximately 30 and 20 kDa, respectively), but cytochrome b 6 has a molecular mass of only 23 kDa in the b6f complex and there is an additional subunit, known as subunit IV, of 17 kDa that is present in this complex. H o w e v e r , when the amino acid sequences of cytochrome b 6 and subunit IV from the plant cytochrome b 6 f complex were compared with the sequences of cytochrome b from the bacterial complex, it became apparent that the cytochrome b gene had been split into two segments to give rise to cytochrome b 6 and subunit IV. The N-terminus of cytochrome b corresponds to the cytochrome b 6 portion of the molecule while the C-terminus corresponds to subunit IV. However, in the case of the cytochrome b6f complex, both b-type hemes are bound to cytochrome b 6 and subunit IV contains no bound electron carriers, although this subunit has been proposed to play a role in quinone binding by the b6f complex (Doyle et al. 1989). Table 2 summarizes the molecular masses of the subunits of the photo-

Table 2. Subunit composition of cytochrome b-c complexes

c-type cytochrome b-type cytochrome Rieske Fe-S Other subunits - core protein i - core protein 2 low mol. wt.

Beef heart mitochondria

Yeast mitochondria

31 31b 42a 24

Subunit Molecular Weight (kD) 31 33 30 26-31 b 42 23 43b 23 20 18

49 46 12 10 8

47 41 16 14 11 8

Spinach

Rps. viridis

17

a Values derived from amino acid sequences derived from DNA sequences. b Values estimated from migration on SDS gels.

Rps. sphaeroides

R. rubrum

Rb. capsulatus

33

30

31 49a

43b 20

36b 17

42 b

13

23

20

124 b6

~

f

Fe S

c

b6f-COMPLEX

Fig. 1. Model describing the membrane topography and interaction of subunits in the chloroplast cytochrome b6f complex. This model uses the hydropathy profile for cytochrome b 6 that predicts 5 transmembrane spanning helices. From Hauska (1986).

Halphen et al. 1991). A model describing the membrane topography of the cytochrome b6f complex from higher plants that is based on hydropathy profiles of the individual subunits presented by Hauska (Hauska 1986) is shown in Fig. 1.

3. The distribution of the cytochrome complex

bCl/b6f

While the cytochrome bc 1 complex was first identified and characterized in the mitochondrial respiratory chain in the 1960s, it was not until the 1980s that it was realized that photosynthetic organisms, both prokaryotes and eukaryotes, contain a similar complex (Hurt and Hauska 1981, Krinner et al. 1982, Gabellini et al. 1982). Because of the unusual properties of the Rieske iron-sulfur center, this is probably the most diagnostic component indicating the presence of the complex. In the case of mitochondria, an E P R signal associated with the Rieske center was first identified by Rieske and co-workers in 1964 (Rieske et al. 1964), but the analogous signal was not identified in chloroplast membranes until 1975 (Malkin and Aparicio 1975) and in photosynthetic bacterial membranes until 1973 (Prince et al. 1975). A similar signal has

been detected in a number of other bacteria, including Paracoccus (Yang and Trumpower 1986) and Bacillus (Lewis et al. 1981, Kutoh and Sone 1988) although aerobically-grown E. coli has been found to show no Rieske iron-sulfur center using similar techniques (Ingledew et al. 1980). While the EPR signal of the Rieske center is certainly diagnostic for the presence of the complex in an organism, it was the pioneering work of Hurt and Hauska that proved beyond a doubt that this type of complex was present in a number of photosynthetic organisms and functioned in a manner similar to the complex in mitochondria. This was accomplished by the isolation and characterization of quinol/cytochrome c (plastocyanin) oxidoreductase complexes from the membranes of chloroplasts (Hurt and Hauska 1981) cyanobacteria (Krinner et al. 1982) and photosynthetic bacteria (Oabellini et al. 1982) The activities and composition of these complexes led to the concept that the cytochrome bcl/ b6f complex is an almost universal component in energy-transducing membranes.

4. Functional considerations of the cytochrome complex The major mechanistic question related to the activity of the cytochrome complex relates to how electron transfer through the complex is coupled to proton translocation across the membrane. Relevant to this question is the observed stoichiometry of 2H ÷ translocated for every electron transferred to an acceptor molecule (Hurt et al. 1983). One additional property of the cytochrome complex that relates to this activity is the observation of 'oxidant-induced reduction' of cytochrome b that has been observed with cytochrome complexes (Erecinska et al. 1972, Hurt and Hauska 1982b). This effect consists of the observation of the transient reduction of cytochrome b after the addition of an oxidant, a result that disagrees with our basic chemical intuition since an electron transfer component is undergoing the opposite reaction from what is expected, that is, cytochrome b is being reduced after the addition of an oxidizing agent. The model that is now the most widely accep-

125 ted that describes the activity of the cytochrome complex is the so-called 'Q-cycle' first proposed by Mitchell in 1975- 76 to explain the anomalous observations made on the complex (Mitchell 1975, 1976). This model envisions two quinone binding sites on the complex (Fig. 2), on opposite sides of the membrane. One of these sites is a quinol oxidizing site (Qo) while the second is a quinone reducing site (Qr)- In the case of the chloroplast membrane cytochrome b6f complex, the Qo site is near the lumenal or inner membrane surface while the Qr site is near the outer or stromal surface of the membrane. At the Qo site, quinol is oxidized in two discrete steps, with the first electron released being transferred to the high potential Rieske iron-sulfur center and the second to one of the two b-type hemes that is located at the Qo site. The electron transferred to the Rieske center is then passed on to cytochrome f, the other high potential electron carrier in the complex, and, ultimately, to the terminal electron acceptor, the soluble protein, plastocyanin. The second electron, which had been transferred to a b-type heme, is transferred across the membrane to the second b-type heme which is located at the Qr site. This electron is then used to reduce a quinone molecule to a semiquinone at this site and by a second round of this cycle, a second electron at the Qr site can reduce the semiquinone to the fully reduced state. The result of two turns of this cycle is that two molecules of quinol have been oxidized, one molecule of quinone has been reduced, two electrons have been passed to plastocyanin and four

2H +

2H+

Fig. 2. The Q-cyclemechanismthat describeselectrontransfer and proton translocation linked to oxidation of plastoquinol and reduction of plastocyanin(PC) by the chloroplast cytochrome b6f complex.

electrons have been removed from quinol and deposited in the lumenal space. Thus, the model provides a clean answer for the confusing stoichiometry of protons and electrons that is observed. In terms of explaining the oxidantinduced reduction of cytochrome b, the model explains this observation by showing that the two electrons in the quinol molecule at the Qo site are treated differently - one is used to reduce the high potential carriers while the second is used to reduce cytochrome b. Thus, oxidation of the high potential carriers by a terminal oxidant leads to a pulling of the electrons from quinol, with one of these electrons being used for the reduction of cytochrome b via the oxidantinduced pathway. Although this mechanism may not fully explain all the observations that have been made with cytochrome complexes, it has served as a useful basis to consider mechanistic aspects of this complex and offers an explanation of diverse results from a number of sources (Rich 1986). 4.1. Molecular biological studies of the

cytochrome bcJb6f complex A. Organization of the genes for the cytochrome complex In some photosynthetic bacteria as well as in Paracoccus denitrificans, the genes encoding the three subunits of the cytochrome bc a complex are organized into a single operon, the fbc operon (Kurowski and Ludwig 1987, Daldal et al. 1987). The three genes for the proteins have been designated fbcF for the Rieske iron-sulfur protein, fbcB for cytochrome b and fbcC for cytochrome c a. As described above, in the case of the photosynthetic bacteria, the question has been raised as to whether an additional low molecular weight subunit is an intrinsic component of the cytochrome complex and while this question is not yet resolved, it is clear that in at least one case, the gene for this additional subunit is not part of the fbc operon. In contrast to this rather simple organization found in many bacteria, the organization of the genes for the cytochrome bc a complex of Bradyrhizobium japonicum is unusual (Thony-Meyer et al. 1989). Only two genes, denoted fbcF and fbcH, encode the three redox proteins. The first

126 of these genes encodes a protein that appears to be homologous to the Rieske iron-sulfur protein while the second gene, fbcH, codes for a high molecular weight protein of approximately 75 000. The N-terminal portion of this protein was homologous to cytochrome b while the Cterminal portion was homologous to cytochrome c a from other organisms. Although a single gene encodes cytochromes b and c~ in this organism, it is clear that two lower molecular weight proteins are the products of the gene, presumably through post-translational processing by a protease that cleaves the high molecular weight product. When one turns to the cyanobacteria and the genes for the cytochrome b6f complex found in these organisms, a different gene organization has been found for the four protein products (Kallas et al. 1988a,b). The genes for these subunits are organized into two operons, denoted petCA and petBD, in the cyanobacterium, Nostoc. The first of these encodes the Rieske iron-sulfur protein and cytochrome f while the second encodes cytochrome b 6 and subunit IV. An operon coding for petCA has also been identified in the cyanobacterium, Synechococcus (Widger 1991). Thus, it appears photosynthetic prokaryotic oxygen evolving organisms have an organization of the genes for the cytochrome complex which in some ways resembles the organization in other prokaryotic organisms, but this organization is clearly more complex than that found in cytochrome bc~-containing prokaryotes. In eukaryotic organisms, the organization of the genes for the cytochrome bc1 and b6f complex reveal a different complexity. As a general rule, one always finds both nuclear and organelle-encoded subunits. Thus, for the mitochondrial cytochrome bc 1 complex, only the cytochrome b gene is found in the organelle genome and all the remaining subunits are nuclear-encoded (Nobrega and Tzagoloff 1980). In the case of the chloroplast cytochrome b6f complex, three of the subunits, cytochrome f, cytochrome b 6 and subunit IV, are chloroplastencoded and the gene for the Rieske iron-sulfur protein is found in the nuclear genome (Willey and Gray 1988). The cytochrome b 6 and subunit IV genes are co-transcribed in chloroplasts but

these two genes are not linked to the gene for cytochrome f. The latter is located in a different operon that is approximately 20 kb away from the petBD operon in the spinach chloroplast genome. Recently, the gene for a putative fifth subunit of the chloroplast cytochrome b6f complex, denoted petE, which encodes for a low molecular weight subunit, has been also found in the chloroplast genome (Haley and Bogorad 1989). The studies of the organization of the genes for the cytochrome complexes in both prokaryotes and eukaryotes have shown a variety of organizational patterns which, in some cases, have aided in a better understanding of the biogenesis of the complex. For example, in most prokaryotic organisms, the simple organization of a single operon that encodes the three individual subunits of the complexes leads to a simple mechanism by which stoichiometric amounts of each subunit in the complex may be produced. The situation in eukaryotic organisms, where both the nuclear and organelle genomes must cooperate in producing the subunits of the complex, presents a problem of complexity that has yet to be understood at the molecular level.

B. Amino acid sequences derived from gene sequences for the components of the cytochrome b6f complex 1. Cytochrome f Cytochrome f sequences are available for a number of organisms that contain a cytochrome b6f complex, including both prokaryotic and eukaryotic organisms (Hauska et al. 1988). For spinach chloroplast cytochrome f, the mature protein contains 285 amino acids with a molecular weight of 31 300. The heme binding domain in this protein has been localized to amino acid residues 56-60: C - X - Y - C - H . As previously mentioned, hydropathy analyses have predicted a single transmembrane span (residues 251-270), and this sequence is found near the C-terminus of the subunit. The heme group and most of the polar amino acids are localized on the lumenal side of the membrane and this allows for interaction with the electron acceptor for the complex, plastocyanin, which is known to be located in the chloroplast lumen. A comparison of the amino acid sequence of

127 Sc

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VT -W

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37

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82

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VrTNT s A ~[TVlGND~ls - -~ v G ~ ~ v ~ A v ~.I~VF-~ PI~.~.~[~M I IQ L K]Q V]L A[_~N[G]K RIKIE G TGL N V G A V L]I PF]P~L~ t ~ _ ~ .~~

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K[_y_]SIEIITIF P I LIAIPIDIP]

..... TIRIV G D G M G - - P D L S V MA ..... P N E Q A A~R]A A N Q G A L P P D L S L I V .....

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R [ K ~ R L G~__~K T V I I L S S]L~V L L S I W V -]K K~F K W A G I K Q R K F V F N P P K P + + +

Fig. 3. Comparison of amino acid sequences of cytochrome c~ and cytochrome f from cytochrome bc~ and b6f complexes. The sequences shown are from Nostoc (Nos), spinach (sp), R. capsulatus (R c) and S. cerevisiae (S c). The arrowhead indicates the start sites of the mature proteins. Boxes surround identical and conserved residues. The C - X - X - C - H heme ligands are overlined and putative m e m b r a n e spanning hydrophobic regions are underlined. Conserved positively charged residues flanking the latter are also indicated. From Kallas et ah (1988b).

cytochrome f with the analogous protein of the cytochrome bc 1 complex, cytochrome ci, shows only a small degree of homology (Fig. 3). There is strict conservation of the heme binding region and in the region of the transmembrane helix, but other portions of the peptides are not highly homologous. Hauska et al. (1988) have calculated only a 10-15% identity between the mitochondrial type cytochrome c 1 and the chloroplast type cytochrome f while there is an approximately 80% identity of residues among different cytochrome f proteins from a variety of oxygenevolving organisms.

2. The Rieske iron-sulfur protein The Rieske iron-sulfur protein is the only well established component of the chloroplast cyto-

chrome b 6 f complex that is nuclear-encoded. The first complete amino acid sequence of this protein was reported by Steppuhn et al. (1987) from spinach chloroplasts and was based on analysis of a cDNA. The mature spinach protein contains 179 amino acids and has a molecular weight of 18 800. The gene for the Rieske ironsulfur protein from the cyanobacterium, Nostoc, has also been sequenced and has been found to encode for a protein of molecular weight 19 200 that is approximately 60% identical in sequence to the spinach chloroplast protein (Kallas et al. 1988b). Analysis of the sequences of the Riekse ironsulfur protein from both cytochrome bc 1 and b 6 f complexes has revealed a number of interesting features (Fig. 4). In general, there is little

128 1

54

sp Rc Pd S c

IAI-TISIIPIADIN[VPDMIQK~ETL~qLLILLIGIAILSILPTGYMILIL~JYASFIFVPPGIGIGAGIT~GT I MSHAEDNAG . . . . TIRIR . . . . . . . . . . . . . . . . . . . . DFL-YHATAATGVVVTGA MSHADEHAGDHGATIRIR .................... DFL YYATAGAGTVAAGA KSTY---IRITPNFDDVLKENNDADKG---RSYA YFMVGAMGLLS SAG Nc GSSSS TFE SPFKGESKAAIKp/P DFGKYMSKAP P STNM .... LF-SYFMVGTMGAI TAAG beef SHTD I KVPDFSDY---IRIRPEVLDSTKSSKESSEA--RKGF SYLVTAT TTVGVAYA

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A~T ~

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A P L NILIEIIIP A Y E m - D G

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DFR

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s G R IIRIK G p A e L NILIEIIle L y E ~ p ~. ~. G KILN X G

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Comparisonof amino acid sequences of the Rieske Fe-S protein from cytochromebc 1 and b6fcomplexes. The sequences shown are from Nostoc (Nos), spinach (sp), R. capsulatus (R c) P. dentrificans (P d), S. cerevisiae (S c), N. crassa (N c) and beef heart (beef). Boxes show identical or conservatively replaced amino acids. Two highly conserved regions, each containing Fig. 4.

probable cysteine and histidine ligands, are overlined. From Kallas et al. (1988b). homology and conservation at the N-terminal portion of these proteins, at least up to amino acid residue 100. There is also little homology or conservation at the extreme C-terminus. There are, however, two regions near the C-terminus of all Rieske proteins that are strictly conserved: firstly is a sequence C - T - H - L - G - C - V and this is followed after an additional 14 residues by another strictly conserved sequence of C - P - C H - G - S . It was originally believed that the four cysteine residues that are found in these two sequences served as the ligands for the for 2Fe2S cluster that is found in all Rieske iron-sulfur proteins. However, spectroscopic results on a number of different Rieske proteins has provided conclusive evidence that this iron-sulfur cluster most likely has two nitrogen ligands

(Cline et al. 1985, Telser et al. 1987, Britt et al. 1991), and the presence of N-ligands has been used to explain the unusually high midpoint oxidation-reduction potential of the Rieske center in comparison with the ferredoxins, which are known to contain S-ligands provided by cysteine residues. This raises the interesting problem of which N-groups in these conserved sequences serve as the ligands for the F e - S cluster and how these are organized with two cysteine ligands. It is significant that the His-129 is found in all Rieske iron-sulfur proteins sequenced to date, but that His-132, found in the mitochondrialtype Rieske center, is absent from the center in spinach and N o s t o c . This analysis would argue that one cysteine and one histidine residue from each conserved peptide unit functions in ligand-

129 ing the Fe-S cluster and using the numbering for the Nostoc protein, a probable combination would be His-110 and Cys-113 from one conserved peptide and Cys-126 and His-129 from the second conserved peptide region. It should also be mentioned that in the general region of these two conserved peptides there are also a number of conserved positively charged amino acids, arginine and lysine, that could also provide Nligands for the Fe-S cluster in these proteins. As briefly mentioned, hydropathy analyses of the sequence for the Rieske protein from spinach and Nostoc has indicated that a single transmembrane helix is likely to be present (Kallas et al. 1988b). This sequence is near the N-terminus and the presumed Fe-S binding domains are near the C-terminus of the proteins. However, similar predictions for the mitochondrial Rieske protein have predicted two transmembrane spanning regions, rather than one (Schagger et al. 1987). What is clear from biochemical experiments is that the Rieske protein can be resolved from the intact cytochrome bc 1 or b6f complex more easily than any other subunit and this may be accomplished in a manner that allows reconstitution of this subunit to the depleted complex (Trumpower et al. 1980, Adam and Malkin 1987). Thus, the integral nature of this subunit with respect to the membrane and with respect to other components of the complex is still an unresolved issue.

3. Cytochrome b 6 and subunit IV As previously described, in the mitochondrial cytochrome bc 1 complex, cytochrome b is present as a single polypeptide containing approximately 380 amino acids, corresponding to a molecular weight of approximately 42 000. In the case of the chloroplast cytochrome b6f complex, this single subunit has been apparently split into two, corresponding to an N-terminal cytochrome b 6 and a C-terminal subunit IV. Extensive analyses of amino acid sequences of a number of c y t o c h r o m e b 6 and subunit IV sequences have confirmed the general nature of the conclusion that has been presented above. C y t o c h r o m e b 6 from oxygen-evolving organisms has a molecular weight of approximately 24 000 while subunit IV has a molecular weight

of approximately 17000. A comparison of a number of cytochrome b6f from photosynthetic organisms reveals a high degree of conservation in these subunits (Fig. 5). Even when the subunit from spinach chloroplasts is compared with the subunit from the cyanobacterium Nostoc, protein sequences are over 90% identical. Similarly, for subunit IV, the protein identity level is 88%. In comparison with the mitochondrial b-type cytochrome, the N-terminus of cytochrome b 6 and the C-terminus of subunit IV show approximately a 40% identity at the protein level. A topic of great controversy concerned the topographic organization of the cytochrome b (b 6 + subunit IV) subunit in the membrane. An original analysis by Widger et al. (1984) predicted that cytochrome b contained 9 transmembrane spanning helices, five of which are localized to cytochrome b 6. Because subunit IV is truncated relative to its mitochondrial counterpart, only three transmembrane helices have been proposed for this subunit so that cytochrome b 6 + subunit IV was predicted to have only 8 transmembrane spanning helices. However, further analyses of the mitochondrial-type cytochrome b molecule in mutants of yeast (Di Rago and Colson 1988, Di Rago et al. 1989) and photosynthetic bacteria (Daldal et al. 1989) that are resistant to specific inhibitors of the complex have recently led to a revised model for this molecule since this original model did not lead to a clustering of inhibitor binding sites as predicted by the Q-cycle model in which the Qo and Qr sites are located on opposite sides of the membrane. It is now generally accepted that the cytochrome b molecule contains 8 transmembrane helices and that helix 4, originally believed to be in the membrane, is at the membrane surface. In terms of the cytochrome b 6 + subunit IV situation, this would imply that cytochrome b 6 has 4 transmembrane helices (Szczepaniak and Cramer 1990). A schematic presentation of the cytochrome b 6 + subunit IV structure showing the currently accepted transmembrane helices is shown in Fig. 6. While there is biochemical evidence that supports this new model (Szczepaniak and Cramer 1990), a detailed structure is still not available although one can definitely conclude on the basis of biochemical experiments that the cytochrome b 6 and subunit IV

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Fig. 5. Comparison of amino acid sequences of cytochrome b 6 and subunit IV of the cytochrome b6fcomplex and cytochrome b of the cytochrome bc 1 complex. Sequences shown are for Nostoc (Nos), spinach (sp), R. capsulatus (R c) and S. cerevisiae (S c).

The top line, labeled sp P, displays the amino terminal sequence of spinach cytochrome b 6 and subunit IV determined by direct amino acid sequencing (see Hauska et al. 1988). Boxes surround identical or conserved amino acids. Conserved His residues, the putative heme-binding ligands, are overlined. Hydrophobic stretches of amino acids that could form transmembrane spanning helices are underlined and numbered with Roman numerals. Their ends are marked with C (= cytoplasmic or stromal) or I (= intrathylakoidal or lumenal) to indicate their possible orientation with respect to the membrane. Solid squares indicate sites with cytochrome b proteins to which mutations have been mapped that confer resistance to quinone-reduction inhibitors. From Kallas et al. (1988a).

subunits are the m o s t h y d r o p h o b i c c o m p o n e n t s of the c y t o c h r o m e b 6 f c o m p l e x . Spectral analyses of b-type c y t o c h r o m e s in c y t o c h r o m e bc 1 and b 6 f c o m p l e x e s indicated the p r e s e n c e of histidine residues as h e m e ligands in b o t h the distal and proximal positions (Carter et al. 1981, S i m p k i n et al. 1989). Since c y t o c h r o m e

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131

intrathylakoidal Fig. 6. Model of cytochrome b 6 and subunit IV showing transmembrane helices and the presumed folding pattern of the subunits in the chloroplast membrane. This model is based on an analysis that predicts only four transmembrane helices for cytochrome b~. Darkened amino acids are sites which have been identified on the basis of inhibitor-insensitivemutants (Qr) in the analogous cytochrome b molecule from yeast or photosynthetic bacteria while heavily outlined residues represent similar amino acids from Qo-inhibitor-insensitive mutants. This figure is courtesy of Dr T. Kallas.

the putative ligands of the two heme groups in the protein. It is interesting to note that subunit IV in the chloroplast complex contains no conserved histidine residues and therefore has not been postulated to be involved in heine binding. The location of the histidine residues within the cytochrome b molecule has led to an arrangement in which two pairs of residues serve as ligands and these two pairs are positioned on opposite sides of the membrane, as shown in Fig. 7. This arrangement could allow for two different regions of quinone binding on the two sides of the membrane, corresponding to the Q~ and Qo sites originally proposed in the Q-cycle mechanism of Mitchell. One feature of mitochondrial cytochrome b relative to chloroplast cytochrome b 6 that remains to be explained is the difference in spectral properties that is observed for the mitochondrial-type b-cytochrome that is apparently absent in the case of cytochrome b 6. How-

ever, recent experiments with chloroplast membranes have now identified small spectral differences between the two chloroplast b-heroes (Baroli et al. 1991) and thus explanations based on subtle differences in sequence may not be valid. A comparison of the amino acid sequences of the b-type cytochromes from a number of sources has revealed few regions of high conservation. The two proposed transmembrane helices that contain the four histidine residues proposed as ligands of the two b-hemes are highly conserved in the over 20 proteins for which sequences are available. The only other region of the protein that shows strict conservation is in the region of amino acids 270-290 in cytochrome b, corresponding to residues 77-80, in subunit IV of the chloroplast complex. Four amino acids, P - E - W - Y , are strictly conserved in almost all sequences that are available for-cyto-

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chrome b or cytochrome b6/subunit IV. In only one sequence to date, the cytochrome b protein from trypanosomes, is there a slight variation in these four amino acids, with a replacement of the terminal tyrosine with phenylalanine, but in all other cases, these amino acids are identical. This strict conservation has led to the proposal that this region plays a critical role in cytochrome b function. Based on hydropathy analyses of this region, it is likely the PEWY sequence forms part of the quinol oxidase site (Qo) and is involved in the binding of the quinol to the cytochrome bca/b6f complex.

4. Evolutionary considerations of the cytochrome bcl/b6f complex The cytochrome bq/b6f complex is almost ubiquitous in energy-transducing membranes, with E. coli the only organism known not to contain this complex. A most interesting feature of this type of complex is its essentially invariant composition as regards electron transfer carriers - a l l energy-transducing cytochrome complexes contain a b-type cytochrome with two hemes, a

c-type cytochrome that contains a single heme and a high-potential iron-sulfur center that contains a 2Fe-2S center. The absence of variations on this basic scheme suggests the cytochrome bcl/b6f complex has undergone minimal change over the time course of the evolution of living organisms and therefore little information can be extracted from the existing organisms on when this type of electron transfer complex appeared in the evolutionary scale. Although the overall components of the cytochrome complexes are quite consistent, differences are apparent when one examines the details of the specific electron transfer proteins within this group of complexes. The most obvious distinction relates to the cytochrome b molecule where in the case of the mitochondrial-type c y t o c h r o m e b c 1 complex, a single high molecular weight subunit is present and is encoded for by a single gene while in the chloroplast-type cytochrome b6f complex, this single protein and single gene has been replaced by two genes encoding cytochrome b 6 and subunit IV. It is not known if the single gene for cytochrome b was

133 split into two genes and that the cytochrome bc~ complex preceded the cytochrome b6f complex on an evolutionary scale or rather the two single genes for cytochrome b 6 and subunit IV were merged to produce the single higher molecular weight cytochrome b subunit. In this regard, investigations of organisms that are considered to be relatively primitive on an evolutionary scale, such as the green photosynthetic bacteria, Chloroflexus and Chlorobium, would be particularly instructive. Resolved complexes from these organisms have not yet been isolated and characterized but studies with membrane fragments have identified components of the complexes, such as the Rieske iron-sulfur protein (Knaff and Malkin 1976, Zannoni and Ingledew 1985, Wynn et al. 1987) and there is a high degree of certainty that these organisms contain, either a bc~ or b6f-type cytochrome complex. In relation to this earlier work with Chlorobium, electron transfer through the membrane-bound cytochrome complex has been reported to be sensitive to antimycin A (Knaff and Buchanan 1975), and this pattern of inhibition has been correlated with the presence of a cytochrome bca complex. In general, it has been found that cytochrome complexes of the bc-type, which contain a single cytochrome b subunit, have an electron transport pathway that is sensitive to antimycin A, while complexes of the b6f-type, which contain the cytochrome b 6 + subunit IV peptides, are not sensitive to this inhibitor (Cramer et al. 1987, Rieske 1976, Hurt and Hauska 1981). It would also be of interest to obtain detailed amino acid sequences of the protein subunits from Bacillus since an earlier report has led to the conclusion that this species may contain a b6f-type cytochrome complex rather than a bcl-type complex more commonly found in non-photosynthetic prokaryotic organisms (Kutoh and Sone 1988). A comparison of the amino acid sequences of the individual subunits of the cytochrome bcl/ b6f complex from a large number of sources shows remarkably few regions of strict conservation considering that these proteins have almost identical functions (Hauska et al. 1988, Howell 1989). In the case of cytochrome cl/f, the only region that shows strong conservation is the heine binding region, and a similar conclusion can be drawn from the sequence of the Rieske

iron-sulfur protein where the putative ironsulfur binding regions of the protein are strictly conserved but other domains are highly variable. For cytochrome b/b6/subunit IV, where the largest numbers of sequences are available, this pattern is maintained with the heme binding helices showing strongly conserved sequences and the PEWY region, presumed to be involved in quinol binding, the most highly conserved sequence in this protein. Other general regions of all these subunits show approximately a 30% conservation, indicating a divergence in structure in these less defined regions. Howell (1989) has identified five regions in the cytochrome b molecule that appear to be consistently conserved during the evolution of this protein, and these regions generally agree with regions that have been found to be associated with increased resistance to cytochrome complex inhibitors, such as antimycin, myxothiazole and stigmatellin. It would then be expected that these regions play important roles in the function of the cytochrome b molecule, but, unfortunately, this type of analysis does not provide details relating to the evolution of the molecule. It is only when interesting variants in structure and function of the components of the complex beyond those that have been studied to date are identified that we may begin to understand the origin of this complex in living systems. It will be of interest to correlate the evolutionary direction of the cytochrome complex along with other photosynthetic complexes, such as the photochemical reaction center and the ATP synthetase, to attempt to understand the principles that led to the establishment of biological membranes as key structures in cellular energy transduction.

Acknowledgement Work from the author's laboratory was supported in part by a grant from the National Institutes of Health.

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Cytochrome bc 1 and b 6 f complexes of photosynthetic membranes.

All photosynthetic membranes contain a cytochrome bc 1 or b 6 f complex that catalyzes the oxidation of quinols and the reduction of a high-potential ...
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