The cytochrome bc 1 complex and its homologue the b 6 f complex: similarities and differences

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1 Photosynthesis Research 79: 25 44, Kluwer Academic Publishers. Printed in the Netherlands. 25 Review The cytochrome bc 1 complex and its homologue the b 6 f complex: similarities and differences Elisabeth Darrouzet 1, Jason W. Cooley 2 & Fevzi Daldal 2, 1 Service de Biochimie Post-génomique et Toxicologie Nucléaire, DIEP, DSV, CEA VALRHO, Bagnols sur Cèze, France; 2 Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104, USA; Author for correspondence ( fdaldal@sas.upenn.edu; fax: ) Received 8 April 2003; accepted in revised form 26 September 2003 Key words: cytochrome bc 1 and b 6 f complexes, electron transfer, proton translocation, respiration and photosynthesis, ubihydroquinone:cytochrome c and plastohydroquinone:plastocyanin oxidoreductases Abstract The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc 1 complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc 1 complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b 6 f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the mitochondrial-type bc 1 complex and the chloroplast-type b 6 f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b 6 f complex. Abbreviations: [2Fe2S] two iron two sulfur cluster; b H high potential b-type heme; b L low potential b-type heme; cyt cytochrome; DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; DCCD dicyclohexylcarbodiimide; DNP-INT dinitrophenyl ether of iodonitrothymol; E m redox midpoint potential at ph 7; EPR electron paramagnetic resonance spectroscopy; HQNO heptylhydroxyquinoline-n-oxide; NQNO nonylhydroxyquinoline-n-oxide; Q quinone (mainly ubiquinone or plastoquinone); QH 2 hydroquinone (mainly ubihydroquinone or plastohydroquinone); Q i site quinone reduction site; Q o site hydroquinone oxidation site; UHDBT undecylhydroxydioxobenzothiazole Introduction The ubihydroquinone:cytochrome (cyt) c oxidoreductase (also called complex III, or bc 1 complex), is a multi subunit enzyme encountered in a very broad variety of organisms such as eukaryotes (uni or multi cellular like yeast or mammals), plants (in their mitochondria), or bacteria (for reviews see Gennis et al. 1993; Gray and Daldal 1995; Crofts and Berry 1998; Yu 1999; Berry et al. 2000). Various forms of the enzyme encountered in these organisms belong to the superfamily of bc complexes (Figure 1) (Yu et al. 1995; Brugna et al. 1998; Xiong et al. 1998; Berry et al. 2000; Schutz et al. 2000; Niebisch and Bott 2003). While mitochondria and bacteria have a bc 1 complex, plant and algal chloroplasts as well as cyanobacteria contain a homologous complex called plastohydroquinone:plastocyanin

2 26 Figure 1. Genetic organization of the structural genes (petabc) for the major subunits of the bc 1 and b 6 f complexes from various organisms. Selected examples from organisms that deviate from the commonly encountered petabc cluster in various bacteria are shown along with the corresponding gene organization seen in mitochondria and chloroplasts. In all cases, the three structural genes corresponding to the Fe S subunit (peta), cyt b (petb)andcytc 1 (petc) are labeled A, B and C and shown in white, gray and black, respectively. The duplicated genes are designated by subscripts, and fused genes are shown separated by dotted lines. H and CC correspond to cyt b fusedtocytc 1 and cyt c 1 fused to cyt c, respectively.

3 27 oxidoreductase or b 6 f complex (for review see Cramer et al. 1996). The bc 1 and b 6 f complexes are key components of energy transducing systems, and are located in the inner mitochondrial membrane, in the thylakoid membrane, or in the cytoplasmic membrane of bacteria. They transfer electrons from a hydroquinone derivative (QH 2 ) (usually ubihydroquinone, menahydroquinone or plastohydroquinone), to a c type cyt, a high potential iron sulfur protein (HiPIP) or a plastocyanin. In doing so, they play a critical role in respiratory and photosynthetic electron transfers events, primarily by contributing to the generation of the electrochemical potential subsequently used by the ATP synthase (Mitchell 1961; Saraste 1999; Dutton et al. 2000). The focus of this review is the comparison of the bc 1 complex (mitochondrial and bacterial-type) and the b 6 f complex (chloroplast-type) in terms of both structure and function utilizing available as well as emerging information (D. Picot, personal communication; also see Soriano et al. 1999; Berry et al. 2000). Specific topics of discussion include the subunit composition, cofactor assembly, inhibitor sensitivity, proton pumping mechanism, concerted electron transfer and the Fe S subunit large-scale domain movement. Subunit composition, sequence comparisons and cofactors assembly The mitochondrial enzyme is made up of subunits of which one (cyt b) is encoded by the mitochondrial genome and the remainder by the nuclear genome (Iwata et al. 1998; Zhang et al. 1998). Amongst these subunits, only the cyt b, cytc 1 and the Fe S subunit are redox active, and they bear as cofactors two b types hemes, one c type heme and one [2Fe2S] cluster, respectively (Figure 2) (Gray and Daldal 1995; Xia et al. 1997; Iwata et al. 1998; Zhang et al. 1998; Yu 1999; Berry et al. 2000; Hunte et al. 2000). The yeast enzyme closely resembles the mammalian complex with some variations in the length and conformation of loops connecting the various conserved secondary structures (Hunte et al. 2000). The chloroplast b 6 f complex is comprised of 8 9 subunits (4 5 large and 3 4 small subunits) (Whitlegge et al. 2002), and the number of subunits encoded by the chloroplast genome versus the nuclear genome varies depending on the species. For example the PetN subunit is encoded by the plastid genome in higher plants and by the nuclear genome in Chlamydomonas reinhardtii or Volvox (Cramer et al. 1996; Hamel et al. 2000; Zito et al. 2002). In cyanobacteria, the subunit composition is similar with four large and two or three small subunits. The four large subunits are clustered into two operons, one encoding the Fe S subunit and cyt f, and another one the cyt b 6 and subunit IV (Figure 1) (Kallas et al. 1988). In some cyanobacteria like Synechocystis, two additional subunits that are homologous to the Fe S subunit are also found (Schneider et al. 2002). In bacteria, the subunit composition is often limited to only the three redox-active subunits, which are encoded by a single operon (petabc or fbcfbc) (Figure 1) (Gennis et al. 1993; Gray and Daldal 1995; Berry et al. 2000). In some cases, a small additional subunit is encountered in species such as Rhodobacter sphaeroides or Rhodovulum sulfidofilum, and proposed to be functionally homologous to the mitochondrial subunit VII (Montoya et al. 1999; Yu et al. 1999). However, bacterial subunits have no pronounced sequence homology with mitochondrial supernumerary subunits. In other bacteria, multiple variations of the basic organization described above have been seen including a b 6 -type shortened cyt b, a fusion between a part of cyt b and cyt c, a multiheme cyt c 1, and even a hybrid cyt bc-cyt oxidase complex such as those characterized in Sulfolobus acidocaldarius or Corynebacterium glutamicum (Anemüller and Schäfer 1990; Liebl et al. 1992; Sone et al. 1995; Yu et al. 1995; Xiong et al. 1998; Pereira et al. 1999; Niebisch and Bott 2003). In addition, recent genome sequence data has begun to reveal that more than one set of bc complexes may be encoded within the same genome in some species, such as Acidithiobacillus ferrooxidans (Brasseur et al. 2002) (Figure 1). Despite the overall subunit composition variability of the b 6 f and bc 1 complexes, similar redox cofactors are found in all cases. The Fe S subunit is highly homologous (Carrell et al. 1997) and the cyt b subunit has a counterpart in the cyt b 6 and subunit IV in the b 6 f complex [not to be confused with the subunit IV encountered in some bacterial enzymes, as mentioned above and discussed below (Chen et al. 1996)] (Widger et al. 1984; Degli-Esposti et al. 1993). There is no direct structural homologue for cyt c 1 in the b 6 f complexes, although the latter also have a c type cyt subunit, called cyt f of unrelated primary structure (Gabellini 1988; Martinez et al. 1994; Soriano et al. 1999). The bacterial, mitochondrial and most recently chloroplast and cyanobacterial enzymes have been crystallized as

4 28 Figure 2 Figure 3

5 29 dimeric structures, and the bacterial enzyme has been shown to form dimers in solution (Huang et al. 1994; Xia et al. 1997; Iwata et al. 1998; Zhang et al. 1998; Montoya et al. 1999; Breyton 2000a; Hunte et al. 2000; Xiao et al. 2001; Zhang et al. 2003). In the case of the chloroplast b 6 f complex, a specific role has been attributed to the dimerization process during the S1 S2 states transition phenomenon (Heimann et al. 1998; Wollman 2001). While the two subfamilies of complexes have sequence homologies restricted mainly to cyt b/b 6 -suiv and to some extent the Fe S subunit, the enzyme architecture as a whole appears to be relatively similar. Specifically, comparison of structures suggests that the trans membrane domains are similar, but the total cofactor content and the extramembranous organization of the Fe S subunit and cyt f subunits are rather different (Breyton 2000b; D. Picot, personal communication). Figure 2. Dimeric structure of the bc 1 complex and the modified Q cycle mechanism. (A) The structure of the entire bc 1 complex dimer, as determined by Iwata et al. (1998), is shown with the Fe S subunit, cyt c 1 and cyt b subunits in blue, green and yellow, respectively. The core proteins are in magenta and the Fe S subunit leader peptide is in cyan, while the other subunits are shown in gray and the metal containing cofactors in red. (B) The three catalytic subunits (cyt b or b 6 + SuIV, cytc 1 or f and Fe S subunit), the cofactors (hemes b L, b H, c 1 or f and the [2Fe2S] cluster), as well as the active sites (Q o and Q i )ofthebc 1 or b 6 f complexes are shown while the nature of the physiological partner of cyt c 1 or cyt f is not identified. This panel illustrates schematically the Q cycle mechanism with proton and electron transfers shown as orange and black arrows, respectively. The double headed, dotted arrow refers to the mobility of the extrinsic domain of the Fe S subunit, and the arrow with a? indicates the hypothetical electron transfer that bypass cyt f in the b 6 f complex (Fernández-Velasco et al. 2001). Figure 3. Structural view of the Fe S subunit from bovine bc 1 complex and spinach b 6 f complex, and partial sequence alignments from various bc 1 and b 6 f complexes. (A) and (B) represent the structure of the Fe S subunit from bovine (Iwata et al. 1998) and spinach chloroplast (Carrell et al. 1997) colored by structure (helices in pink, sheets in orange, turns in blue). The well-conserved regions of box I and box II have been colored in green with the His ligands in ball and stick. The Cys residues of the boxes are also in ball and stick model and colored in yellow. The proline residue 175 in bovine and 142 in spinach is highlighted in cyan as well as Thr53 in spinach, and Lys73 plus the linker region in bovine. The same color code described above is used in the sequence alignments shown underneath, and the disulfide bridge is indicated. For the sake of simplicity only two regions, the highly conserved cluster domain containing box I and box II, and the functionally important but less conserved linker domain, are shown. Bo, Sc, Rc, Cr, Sp, No, Sy correspond to Bos Taurus, Saccharomyces cerevisiae, Rhodobacter capsulatus, C. reinhardtii, Spinach, Nostoc and Synechocystis sp. PCC6803, respectively. Fe S subunit and its [2Fe2S] cluster The Fe S subunits are extremely well conserved among the family of bc complexes, and their three dimensional structures from both the bc 1 and b 6 f complexes have been solved at atomic scale resolution (Iwata et al. 1996; Carrell et al. 1997; Iwata et al. 1998; Zhang et al. 1998; Hunte et al. 2000). The Fe S protein is about 200 amino acids (196 for the mature bovine protein) in length, with a single trans membrane helix and the amino terminus (N-ter) located in the matrix, stroma, or cytoplasm depending upon the organism (Figure 3). The carboxyl terminus (Cter) forms a membrane-extrinsic domain that protrudes into the mitochondrial inter membrane space, the thylakoid lumen or the bacterial periplasm. Whatever the origin of the Fe S subunit, the signature motif sequences of box I (CTHLGC) and box II (CPCHGS) which provide the two cysteine and two histidine ligands for its high redox potential [2Fe2S] cluster, are conserved (Davidson et al. 1992a; van Doren et al. 1993; Liebl et al. 1997; Link 1999). Higher amino acid conservation from species to species is seen in the region of the Fe S subunit surrounding the metal center, while the remaining parts of the polypeptide are similar only at the structural level (Carrell et al. 1997; Soriano et al. 1999). Illustrating this conservation is the presence in all bc complexes of a disulfide bridge (Figure 3) that is proposed to contribute to the cluster stability (Davidson et al. 1992a; Iwata et al. 1996). Several differences between the Fe S subunits of the bc 1 and b 6 f complexes can be found in the regions where the Fe S subunit interacts with the other subunits, and where the surrounding ph is different depending on the organisms [see Carrell et al. (1997) for a structural comparison]. Notable differences include the bilobal shape of the Fe S subunit from the b 6 f complex versus its single lobed counterpart in the bc 1 complex and the peptide bond between Gly141 and Pro 142 is found in a cis conformation in the spinach b 6 f complex whereas in the bovine bc 1 complex is in trans conformation (Figure 3) (Carrell et al. 1997; Iwata et al. 1998). The significance, if any of this cis/trans configuration is unknown. In addition, the region linking the extrinsic domain and the trans membrane helix of the Fe S protein is longer and rich in glycine residues in the case of the b 6 f complex as compared to its counterpart in mitochondrial or bacterial bc complexes (Yan and Cramer 2003). The redox midpoint potential (E m ) of the [2Fe2S] cluster is similar in mitochondria, chloroplasts and

6 30 Table 1. Characteristics of the different cofactors of the bc 1 and b 6 f complexes from various species Species [2Fe2S] cluster Cyt b L Cyt b H Cyt c 1 or cyt f g x, g y, g z E m7 (mv) α band E m7 (mv) α band E m7 (mv) α band E m7 (mv) λ max (nm) λ max (nm) λ max (nm) Bovine a 2.02, 1.89, and R. capsulatus b 2.02, 1.895, and R. capsulatus b 6 c c R. sphaeroides d 2.03, 1.90, and R. sphaeroides b 6 f like e PS3 f 2.03, 1.90, Spinach b 6 f g 2.03, 1.9, C. reinhardtii h a From Matsuno and Hatefi (1999); Link (1999). b From Robertson et al. (1993); Baymann et al. (1999); Link (1999); Saribas et al. (1999). c From Saribas et al. (1999). d From Kuras et al. (1998); Link (1999). e From Kuras et al. (1998). f From Liebl et al. (1992). g From Link (1999); Kroliczewski and Szczepaniak (2002); Carrell et al. (1997); Kramer and Crofts (1994). h From Pierre et al. (1995); Nitschke et al. (1992); Ponomarev and Cramer (1998). bacteria (around 320 mv, in the latter case) using ubihydroquinone or plastohydroquinone as substrate, but it is much lower in bc complexes oxidizing menahydroquinone, like Bacillus PS3 or Thermus thermophilus (E m s around mV) (Liebl et al. 1992; Link 1999) (Table 1). A similarly low E m (around 135 mv) has also been reported for one of the three Fe S protein homologues of Synechocystis (Schneider et al. 2002). Parameters that affect the E m value of the [2Fe2S] cluster include the nature of the amino acid sequences of the boxes I and II (Davidson et al. 1992a; Liebl et al. 1997), the presence of the disulfide bridge (Davidson et al. 1992a) as well as the hydrogen bond network around the metal center (Denke et al. 1998; Schröter et al. 1998; Guergova-Kuras et al. 2000). In addition, the position of the Fe S subunit in the bc complex and the chemical nature of the occupants (substrate, product and inhibitors) of the hydroquinone oxidation (Q o ) catalytic site of the enzyme also influence the E m (Darrouzet et al. 2002; Shinkarev et al. 2002). The E m of the [2Fe2S] cluster can be readily monitored using electron paramagnetic resonance spectroscopy (EPR). The EPR spectrum of the [2Fe2S] cluster is highly sensitive to its local environment, and in particular the line shape at the g x transition varies in both its position in the magnetic field sweep, and its shape depending on the position of the head domain and the occupant of the Q o site (Ding et al. 1992; Sharp et al. 1998). With Q at the Q o site, the spectra from the bc 1 and b 6 f complexes are centered at a similar magnetic field value (e.g., g y = 1.90), while the g x trough is shifted slightly up field in the latter case (Table 1) ( versus 1.80 for the bc 1 complex). In all bc complexes, both the position and the shape of the g x signal are modified by the presence of inhibitors like stigmatellin, UH- DBT, and DBMIB (Bowyer et al. 1982; von Jagow and Ohnishi 1985; Malkin 1986; Sharp et al. 1999; Roberts and Kramer 2001). However, whether these inhibitors mediate the changes in the EPR spectrum by affecting the [2Fe2S] cluster directly or indirectly is unknown. It has been observed that in the bovine or R. capsulatus enzyme the E m of the cluster increases by about 200 mv in the presence of stigmatellin (von Jagow and Ohnishi 1985; Darrouzet et al. 2002). In contrast, in some bacteria like Bacillus PS3, which contain a b 6 f-like complex (i.e., with a split cyt b), the E m of the cluster remains low while the g x transition changes in response to stigmatellin (Liebl et al. 1992). In the case of the b 6 f complex, DBMIB or stigmatellin do not increase the E m of the cluster but the g x transition shifts to a higher field value and becomes broader (Malkin 1986). Conversely, DBMIB decreases the E m and induces a pk-shift of the redoxlinked proton (Schoepp et al. 1999). One benefit of the different modes of interactions between inhibitors and

7 31 bc complexes is that the varied specificity might allow for the targeting of specific organisms for medicinal or agricultural purposes (Srivastava et al. 1999). The Fe S subunit is thought to be translocated across the membrane via the twin arginine transport (TAT) pathway, as suggested by the presence of a highly conserved RR [or RK as in C. reinhardtii (Molik et al. 2001)] motif present in the signal sequence of the preprotein. As the TAT pathway is considered to be specific for folded proteins, the Fe S subunit may be translocated with the [2Fe2S] cluster already inserted into the apoprotein prior to translocation across the membrane and incorporation into the enzyme complex (Bernhard et al. 2000; Molik et al. 2001). In the case of the bovine mitochondrial bc 1 complex, it has been observed that the signal peptide of the Fe S subunit, although processed during its mitochondrial import, stays associated with the enzyme and constitutes its eleventh subunit (corresponding to subunit nine in the structure) (Zhang et al. 1998). Cyt b subunit and its b-type hemes b L and b H In the mitochondrial-type enzyme, cyt b is an integral membrane protein of about 48 kd, with eight trans membrane helices, of which the first four (helix A to D) form a four-helix bundle structure and bear the two b-type hemes (b L for low potential and b H for high potential b-type heme). The remaining four helices (helix E to H) are devoid of any prosthetic group in the cyt bc 1 (Figure 4). The cyt b subunit has both its N- and C-terminal on the matrix or cytoplasmic side of the membrane, and houses two catalytic domains that bind Q and QH 2 as substrates and products. Of these catalytic domains, the Q i (quinone reduction) site is buried further into the protein and is closer to the matrix or cytoplasmic side of the membrane. Conversely, the Q o (hydroquinone oxidation) site faces the inter membrane space or periplasm in mitochondria or bacteria, respectively and is more surface-exposed at the interface of the Fe S and cyt b subunits (Xia et al. 1997; Iwata et al. 1998; Zhang et al. 1998; Hunte et al. 2000). Excitingly, the forthcoming crystal structure of the b 6 f complex is now revealing a third heme molecule located near the heme b H and the Q i site, and covalently linked to the cyt b 6 subunit (D. Picot, personal communication). This novel heme group appears to be a clear indication of a major difference between the bc and bf complexes. The two hemes, b H and b L, shared by both bc and bf complexes are non-covalently but stereo specifically inserted into the subunit, and are axially coordinated by four histidine residues (H97 and H196 for heme b H and H83 and H182 for heme b L, bovine numbering) located on the second (B) and fourth (D) helices of cyt b. In chloroplasts and some bacteria including cyanobacteria the first four helices (A D) form a separate subunit called cyt b 6 that is homologous in both structure and sequence to the first four helices of cyt b. In these species another subunit called subunit IV contains three membrane-spanning helices homologous to helices E, F and G of cyt b, and no eighth helix corresponding to helix H exists (Widger et al. 1984; Degli-Esposti et al. 1993) (Figure 4). Remarkably, the amino acid sequence identity between the mitochondrial cyt b and the chloroplast cyt b 6 plus subunit IV is extremely high (as high as 60%) suggesting that they might have evolved following appropriate splitting or fusion events of the cyt b or the cyt b 6 and the subunit IV, respectively (Widger et al. 1984; Xiong and Bauer 2002). Previously, it was noted in the case of the cyt b 6 -like subunit of Bacillus subtilis that one of the b hemes remains associated with the denatured protein during SDS-PAGE analysis (Yu and Le Brun 1998), suggesting that it might be covalently linked to the protein. This covalent attachment appears also to be the case for the b 6 f complex from higher plants, algae and cyanobacteria, as some cyt b subunits also retain their heme groups under similar SDS-PAGE conditions (Kuras et al. 1997). However, in vitro reconstitution experiments with spinach cyt b 6 suggest that the b hemes need not be attached covalently (Kroliczewski and Szczepaniak 2002). This long-standing puzzle now seems to be solved due to a third heme moiety that is located at the interface of the cyt b 6 and the subunit IV near the Q i site. This additional heme is covalently ligated via a single thioether bond between the vinyl group of the protoporphyrin IX and a highly conserved cysteine residue (Cys 35 in C. reinhardtii), as revealed by recent crystal structure of algal chloroplast b 6 f complex (D. Picot, personal communication). As expected, the structural differences observed between the bc 1 and b 6 f complexes also reflect on the physicochemical properties of the b-type hemes with respect to their absorption spectra and their E m values. In the bc 1 complex, the b H heme has a α-band centered at 562 nm whereas the b L heme has a split α-band with peaks centered at 558 and 565 nm, probably due to steric constraints on its axial ligands (Salerno 1984). In the case of the b 6 f complex, the b H heme is similar with its α-band centered around 562 nm, but the b L

8 32 Figure 4 Figure 5

9 33 heme does not exhibit a split α-band and has about the same λ max as b H. Moreover, in the case of the bc 1 complex the E m values of the b L and b H hemes are around 90 and +50 mv, respectively, but they are somewhat variable among species (Table 1). On the other hand, the E m values of the two b type hemes in isolated b 6 f complexes are around 160 and 85 mv (Pierre et al. 1995) while those of membraneassociated complexes have b heme E m values closer to each other (Furbacher et al. 1989; but also see Kramer and Crofts 1994). Several hypotheses have been proposed to rationalize the molecular bases of the spectroscopic and thermodynamic differences between these enzymes. For example, it has been considered that physical separation of the four helical bundle part from the remainder of cyt b, as seen with the cyt b 6 of the b 6 f complexes, might decrease the axial constraints on the helices and eliminate the de loop that shields the Q i site and the b H heme. The works of Kuras et al. (1998) and Saribas et al. (1999), in which a b 6 like subunit has been engineered by dividing a bacterial cyt b into two parts, have shown that indeed this splitting can explain some spectral differences, as in these mutants the b L and b H hemes have slightly modified spectra with an α- peak centered at nm. However, with regards to the E m, no change for R. capsulatus b 6 c 1 complex (Saribas et al. 1999), or only a small increase for R. Figure 4. Structural view of cyt b subunit from bovine bc 1 complex, and partial sequence alignments from various bc 1 and b 6 f complexes. Cyt b subunit from bovine bc 1 complex (Iwata et al. 1998) is colored in order to highlight the homologies with cyt b 6 f complex. Regions with no homologies (C-terminal and de loop) are colored in green, the four helices homologous to cyt b 6 are in yellow with the two hemes in red and the His ligands in cyan, and the three helices homologous to subunit IV are in orange. The two portions of cyt b that correspond to cyt b 6 and Su IV are shown separately on the right. The sequence alignments shown underneath the figure is to illustrate the regions of cyt b and cyt b 6 surrounding the His axial ligands of the b H and b L hemes in helices B and D, respectively. Bo, Sc, Rc, Cr, Sp, No, Sy correspond to Bos Taurus, S. cerevisiae, R. capsulatus, C. reinhardtii, Spinach, Nostoc and Synechocystis sp. PCC6803, respectively. Figure 5. Structural view of cyt c 1 and cyt f subunits. Cyt c 1 (A) (Iwata et al. 1998) and cyt f (B) (Martinez et al. 1994) are represented with a surface display and colored according to their electrostatic potential (ViewerLite, Accelrys, Underneath the figure are shown the sequences alignments corresponding to the covalent heme binding regions of both c-type cytochromes that are the only well conserved amino acid sequences. Bo, Sc, Rc, Cr, Sp, No, Sy correspond to Bos Taurus, S. cerevisiae, R. capsulatus, C. reinhardtii, Spinach, Nostoc and Synechocystis sp. PCC6803, respectively. sphaeroides bc 1 complex (Kuras et al. 1998) has been found. Additional complementary hypotheses also originated from the alignments of various cyt b and b 6 sequences. For example, on the cyt b 6 helix D there is an insertion of one amino acid residue between the histidine ligands of the hemes as compared to cyt b (i.e., 14 amino acids between b L and b H hemes instead of 13) (Figure 4) (Widger et al. 1984; Degli-Esposti et al. 1993). Such alignments also highlight unusual amino acid residues that are only conserved in cyt b 6 and not in cyt b. Examples include the Cys 35 in C. reinhardtii mentioned above involved in covalent attachment of the third heme of cyt b 6 (D. Picot, personal communication) or the presence of Arg 87 in Nostoc cyt b 6 that might affect the properties of b-hemes (Cramer et al. 1996; Yu and Le Brun 1998). In this respect, the unusual optical properties of the b H heme (λ max 567 nm) of the colorless alga Polytomella spp. might be the result of a change of the two conserved amino acid residues, Ser36 instead of a non polar residue like Ala, and Leu or Phe, and Tyr114 instead of Trp (Antaramian et al. 1998). The differences between the bc 1 and b 6 f complexes also extend to the Q i and Q o sites of the enzyme. The Q i site environment is obviously different between these enzymes due to the physical separation of cyt b into cyt b 6 and subunit IV and the presence of an additional heme nearby (D. Picot, personal communication). The remarkable differences are reflected by the fact that the b 6 f complex is insensitive to the classical Q i site inhibitors of the bc 1 complex such as antimycin A, HQNO and funiculosin (Hauska et al. 1983; Park and Daldal 1992), but not to NQNO (Jones and Whitmarsh 1986). It has even been proposed that the Q i site of the b 6 f complex might be affected by inhibitors like myxothiazol that are known to be specific for the Q o site in the bc 1 complexes. However, the sensitivity to the Q i site inhibitor antimycin A has not changed drastically in R. capsulatus b 6 c 1 mutant complex that has a cyt b 6 like structure (Saribas et al. 1999). Moreover, there is no detectable stable semiquinone (SQ) in the Q i site of the b 6 f complex, and to what extent this is related to the newly detected third heme group in close proximity is intriguing. Finally, consistent with the overall differences in this region, the conserved His217 (in R. capsulatus numbering), which is very close to the cyt b 6 -subunit IV interface and often implicated in the stability of this SQ at the Q i site of the bc 1 complex is not conserved in cyt b 6 (Gray et al. 1994).

10 34 In the case of the Q o site, the differences seen between the bc 1 and the b 6 f complexes are even more refined than the Q i site, as the latter enzyme is sensitive to class-ii inhibitors like stigmatellin (von Jagow and Link 1986; Nitschke et al. 1989) and UH- DBT, but not to class-i inhibitors like myxothiazol (Rich 1984) or MOA-stilbene. Moreover, the mode of inhibition exerted by the class-ii inhibitors on the b 6 f complexes might be different than that on the bc 1 complexes, as they do not increase the E m of the [2Fe2S] cluster of the Fe S subunit. Alternately, the b 6 f complex is much more sensitive than the bc 1 complexes to DBMIB, which is distinct from both the class-i and class-ii inhibitors as its behavior depends on its redox state (Schoepp et al. 1999). Experiments with DBMIB and DNP-INT also revealed that in some chloroplasts, the Q o site is composed of two sub domains (Barbagallo et al. 1999), as Roberts and Kramer (2001) have found that two molecules of DB- MIB can bind to the Q o site of the b 6 f complex. It is also known that several other molecules (e.g., 3-CHMDB, 3-chloro-5-hydroxyl-2-methyl-6-decyl-1, 4-benzoquinone) also interact with the Q o site of the bc complexes differently as 3-CHMBD inhibits the bc 1 complex, but it is a substrate for the b 6 f complex (Gu et al. 1989). The implication of certain amino acid residues (e.g., M140, F144, G152, G158 R. capsulatus numbering) in specific positions of cyt b as possible molecular bases of inhibitor resistance naturally encountered in various bc complexes has been highlighted by mutant analyses (Daldal et al. 1990; di Rago et al. 1990; Tokito and Daldal 1993; reviewed in Brasseur et al. 1996). Similar experiments were also performed in the cyanobacterium Synechococcus sp. PCC 7002 which can become more DBMIBresistant or myxothiazol-sensitive (Lee et al. 2001). An extreme case with regard to inhibitor interactions has been seen recently with the bc 1 complex of Rubrivivax gelatinosus, which is resistant to stigmatellin, myxothiazol and antimycin A (Ouchane et al. 2002). Undoubtedly, the emerging structures of the b 6 f complexes will shed more light on our understanding of the molecular basis of these observations. Clearly, more remains to be discovered about the interactions of the bc 1 and b 6 f complexes with the Q o and Q i site inhibitors by thorough analyses of currently available genome databases (Kranz et al. 2002) and emerging crystal structures (Zhang et al. 2003) with respect to specific sequence patterns and diffraction patterns associated with inhibitor resistance. Cyt c 1 and cyt f subunits and their c-type hemes Both cyt c 1 and cyt f have been crystallized at high resolution (Martinez et al. 1994; Xia et al. 1997; Iwata et al. 1998; Zhang et al. 1998; Hunte et al. 2000; and for a detailed structural comparison, see Soriano et al. 1999). At first glance, the structures of the cyt c 1 and cyt f subunits appear unrelated although both soluble domains are anchored to the membrane by a similar C-terminal helix, and each domain houses a c-type heme that is reduced by a [2Fe2S] cluster (Figure 5). Perhaps such large structural differences are not unexpected as these two proteins have very different partners with which they interact physically, and to which they transfer an electron: a c-type cyt (cyt c, c 2, c y, c 8 ) or a high potential iron sulfur protein (HiPIP) in the case of the bc 1 complex versus plastocyanin or a c-type cyt (cyt c 6 )inthecaseofthe b 6 f complex. In addition, the ph of the environment in which they perform their specific functions is very different between the mitochondria and chloroplasts. Yet, as they both interact with a c-type cyt they both exhibit surface charges provided by patches of acidic and basic residues, and hydrophobic regions that are important for initial docking of their partners (Axelrod et al. 2002; Lange and Hunte 2002). Interestingly, although multiple variants of the cyt c 1 subunit have been encountered in the bc 1 complexes, for example the fusion of subunit IV and a c-type cyt in B. subtilis (Yu and Le Brun 1998), or the diheme c-type cyts in Heliobacillus mobilis or C. glutamicum (Xiong et al. 1998; Niebisch and Bott 2003), apparently no variant of cyt f has been seen (Figure 1). Thus, evolutionary plasticity of the bc 1 and b 6 f complexes appears different with respect to their c-type heme bearing subunits and their physiological partners. The cyt c 1 and cyt f are also dissimilar with respect to the orientation of the heme group with the former being almost perpendicular to the membrane, and the latter at a angle versus the membrane (Martinez et al. 1994; Berry et al. 2000). In addition, a pathway of fixed water molecules in cyt f that is absent in cyt c 1 has been described (Martinez et al. 1994; Ponomarev and Cramer 1998). Lastly, the distances between the heme-irons of each monomer, as well as the over all positions of the heme domains of the cyt c 1 and cyt f are also different in the bc 1 complex as compared with the b 6 f complex (Breyton 2000b; D. Picot, personal communication). Despite the differences described above between the cyt c 1 and cyt f, they both have their heme

11 35 moiety covalently attached by two cysteine residues of a CxyCH motif located towards the N-ter of the protein (Figure 5). Exceptions to this bis-cysteine ligation have been encountered in some algae, such as Euglena gracilis (Mukai et al. 1989), or in trypanosomes (Priest and Hajduk 1992) and in apicomplexan parasites like malaria where heme is attached to the apoprotein by only one cysteine residue. For the fifth and sixth ligands of the heme moiety, one finds a His-Met in cyt c 1 whereas a His-NH 2 of the N-terminus is used in cyt f (Martinez et al. 1994). Apparently this axial ligation difference contributes to the different EPR signatures of the cyt c 1 and cyt f as these spectra become more similar in cyt c 1 variants in which the Met ligand is substituted by a Lys residue (Li et al. 2002). On the other hand, the E m values of the cyt c 1 and cyt f are very similar in some cases (e.g., around 330 mv in C. reinhardtii and R. capsulatus, and a bit lower in the bovine case) (Table 1), or a bit higher in most higher plants (around 360 mv from mustard, radish and turnip) (Tanaka et al. 1978; Takabe et al. 1980; Metzger et al. 1997). In fact, the E m differences observed between the cyt f and the mitochondrial cyt c 1 were attributed to the presence of the two aromatic residues (the N-ter liganding Tyr and the Phe residue at position 4), which apparently shield the heme moiety from the aqueous phase (Soriano et al. 1999). Interestingly, a unique disulfide bridge that anchors a loop shielding the heme group is present in some bacterial cyt c 1 (e.g., R. capsulatus), and its absence decreases the E m dramatically unless a β-branched amino acid is present two residues away from the heme sixth ligand Met, like in the mitochondrial cyt c 1 (Osyczka et al. 2001). Both cyt c 1 and cyt f play critical roles in to the biogenesis of the bc 1 and b 6 f complexes, by controlling the assembly and stability of these enzymes. In R capsulatus, R sphaeroides, orparacocccus denitrificans, mutants that lack the cyt c 1 subunit for various reasons (e.g., mutants that are defective in cyt-c biogenesis, or those that make a truncated soluble cyt c 1 or those that carry a deletion of it) virtually lack the cyt b and Fe S subunit in their membranes (Konishi et al. 1991; Davidson et al. 1992b). On the other hand, R. capsulatus cyt c 1 (Davidson et al. 1992a, b) or chloroplast cyt f (Kuras and Wollman 1994) are more stable in the membrane in the absence of other subunits, possibly providing a nucleation site for the assembly of the holo-complexes. Moreover, in chloroplasts an additional translational regulation process, called control by epistasy of synthesis, that monitors the presence of the other assembled dominant proteins also operates during the assembly of the b 6 f complex (Choquet et al. 1998). Other subunits or cofactors There is no pronounced homology between the bc 1 and b 6 f complexes that can be detected when the non-catalytic subunits are considered. These species-specific supernumerary subunits, (8 9 in the mitochondrial enzyme and 4 5 in the chloroplast/cyanobacterial enzyme) are important for roles that are not apparently directly related to the QH 2 oxidoreductase activity of the bc complexes. Instead, they often affect the stability or protection of the enzyme in response to their different physiological environments. For example, the core subunits (subunits I and II) of the mitochondrial enzyme are homologous to matrix processing proteases (Glaser and Dessi 1999), while subunit VI of the bc 1 complex from yeast is implicated in its dimerization (Schmitt and Trumpower 1990). In some bacteria, such as R. sphaeroides, a fourth subunit, which apparently contributes to QH 2 binding and stability of the enzyme has been described (Chen et al. 1996; Yu et al. 1999). However, in many other bacterial species including R. capsulatus the corresponding protein does not copurify with the bc 1 complex under routinely used conditions, albeit a putative gene coding for a peptide with high sequence homology is present in their genome sequence (Robertson et al. 1993). In the case of the b 6 f complex, the topology and function of the PetL subunit has been recently assigned using chimeric constructs with subunit IV (Zito et al. 2002). Additionally, a regulatory role in photosynthesis has also been attributed to another subunit, PetM (Schneider et al. 2001). Lastly, the ferredoxin:nadp + oxidoreductase protein is found to copurify with the b 6 f complex of spinach chloroplasts (Zhang et al. 2001). While the significance, if any, of this association is currently unclear, it might have interesting implications in the light of the forthcoming structures of the b 6 f complexes. A structure-based comparison of the shapes of the bc 1 and b 6 f complexes from a viewpoint perpendicular to the membrane reveals that they are markedly different in respect to the location of their supernumerary subunits (D. Picot, personal communication). Whether this shape difference has any relevance in respect to the interaction of the bc 1 and b 6 f complexes with their physiological neighbors in the membranes will certainly be investigated in the coming years. It is

12 36 noteworthy that in addition to the differences in subunit composition, several additional small molecules are also found in various bc 1 and b 6 f complexes. A ß-carotene (Zhang et al. 1999), a chlorophyll (Huang et al. 1994), and five monogalactosyl-diacylglycerol lipids are associated with the b 6 f complex, while a cardiolipin molecule seems absolutely essential for the function of the bovine enzyme (Gomez and Robinson 1999). Recent crystal structures are now defining the precise locations of these additional molecules, and hopefully, will lead to a better understanding of their physiological roles. Mechanism of function The Q cycle mechanism The bc 1 and b 6 f complexes have high potential and low potential redox chains that work in concert, and upon two consecutive turnovers they convert two QH 2 to Q, and one Q to a QH 2, with the net result of a single QH 2 consumption, according to the modified Q cycle mechanism (Figure 2) (Mitchell 1975; Crofts et al. 1983). This mechanism involves the net release of four protons to one side of the energy transducing membrane in association with the oxidation of the two QH 2 molecules. All the while, two protons are removed from the other side of the membrane via the reduction of a Q molecule to a QH 2. In addition to building a ph gradient ( ph), the bc complexes also move electrons from one face of the membrane to the other, leading to charge separation ( ) and membrane energization. This unique and sophisticated mechanism is best described by considering two consecutive turnovers of the enzyme (Figure 2). The first turnover is initiated upon the oxidation of a QH 2 at the Q o site; one electron is conveyed to the high potential redox chain, comprised of the [2Fe2S] cluster and c 1 heme, whereas the other electron is transferred to the low potential chain, formed by the b L and b H hemes. The b H heme reduces a Q molecule bound at the Q i site, yielding a SQ radical intermediate. Upon the oxidation of a second QH 2 at the Q o site, the electron arriving at the Q i site converts the SQ to QH 2.Remarkably, the two electrons from the same QH 2 at the Q o site are conveyed to two opposite sides of the membrane using a concerted mechanism commonly termed the electron bifurcation reaction. Currently, while the overall schemes of electron transfer pathways within the bc 1 and b 6 f complexes are known (Figure 2), the mechanistic details of the bifurcation reaction, which is crucial for the energetic efficiency of these enzymes, are unclear. For example, it is not understood why the second electron emanating from the oxidation of QH 2 follows an apparently thermodynamically less favorable pathway. Over the years, several hypotheses have been formulated to elucidate the sequential events that occur during this concerted reaction, in part to explain why the SQ intermediate of the QH 2 to Q reaction is not readily detected. The double occupancy model invokes a great instability of the SQ formed after electron transfer to the [2Fe2S] cluster and the presence of a second Q molecule closer to heme b L that acts as an immediate acceptor for the second electron at the Q o site (Ding et al. 1995). In the proton gated charge transfer model, two Q molecules are present at the Q o site but a conformational catalytic switch is proposed to forbid the [2Fe2S] cluster from accepting the second electron (Brandt 1996). Other models invoke the formation of stable intermediates between SQ and reduced Fe S subunit (causing the SQ to be nonparamagnetic) until the second electron is transferred to heme b L (Link 1997), or between Q, His161 (bovine numbering) which is one of the [2Fe2S] cluster ligands in the Fe S subunit and Glu272 of cyt b as a third partner (Snyder et al. 2000). Still others propose physical separation of the reactive species such as the rolling over of SQ (from a [2Fe2S]-proximal to a heme b L - proximal position thus changing the distances between the redox partners) (Crofts et al. 1999a, b), or the displacement of the SQ from the Q o site to the Q i site (Joliot and Joliot 1994) or even involvement of dimeric structures of the enzyme (Gopta et al. 1998; Trumpower 2002). To date, in the bc 1 complex there is no clear evidence for physiological non-concerted electron transfer, although it has been reported that the production of O 2 is increased in the presence of inhibitors like antimycin (Muller et al. 2002). On the other hand, b 6 f complex mutants that perturb the water chain in cyt f differently affect the cyt b and cyt f reduction kinetics (Ponomarev and Cramer 1998). However, the altered cyt f kinetics could reflect indirect effects, as the initial concerted mechanism during the QH 2 oxidation should take place between cyt b L and the [2Fe2S] cluster, and not between cyt b L and f or c 1.Moreover, in the b 6 f complex apparently plastocyanin reduction can also bypass at least partially cyt f, and occur directly via the [2Fe2S] cluster, possibly contributing to the differences seen between the cyt f and cyt b L kinetics (Fernández-Velasco et al. 2001).

13 37 Movement of the Fe S subunit during Q o site catalysis The recent resolution at atomic scale of the structure of the mitochondrial bc 1 complex in different species, in the presence or absence of various inhibitors, and in various crystal forms (Iwata et al. 1998; Kim et al. 1998; Zhang et al. 1998; Gao et al. 2002) led to an unprecedented discovery. Comparison of these structures revealed dramatically different conformations for the Fe S subunit, suggesting that a large-scale domain movement of this subunit (57 rotation and 16 Å translation for the [2Fe2S] cluster) may occur during the intra-complex electron transfer (for a review, see Darrouzet et al. 2001). In some of the crystals, the Fe S subunit is close to cyt c 1 (c 1 position), or in a position near the ef loop of cyt b (intermediate position), or in positions at the Q o site (b position, and stigmatellin position) (Iwata et al. 1998; Kim et al. 1998; Zhang et al. 1998; Gao et al. 2002; S. Iwata, personal communication). The complete structure of the b 6 f complex at atomic scale resolution has just recently been accomplished for both algal and cyanobacterial species, and is consistent with the movement of the Fe S protein. In addition, studies with 2D crystals have indicated that the b 6 f complex also undergoes important conformational changes in respect to the location of some trans membrane helices of cyt b (Breyton 2000a, b). Undoubtedly, additional structural information pertinent to this point will become available in the near future. In any event, clearly none of the structural conformations alone can accommodate the overall spectroscopic and kinetic data available. In the structures with the Fe S subunit in the stigmatellin, b or even intermediate positions, the [2Fe2S] cluster is indeed too far away from the c 1 heme to mediate the rapid electron tunneling observed between these cofactors. On the other hand, the EPR signature of the [2Fe2S] indicates a close interaction between this cluster and the occupants of the Q o site (Q or inhibitor), which is incompatible with the large cleft between the [2Fe2S] cluster and the Q o site observed in the structures with the Fe S subunit in the c 1 position. These initial observations suggested that the Fe S subunit moves back and forth during the catalytic cycle of the enzyme, and subsequently, additional data have accumulated to further support the movement. First, EPR spectroscopy of oriented membrane preparations in the presence of various inhibitors or Q or QH 2 indicated that the oxidized and the reduced [2Fe2S] cluster exhibited different orientations within the enzyme (Brugna et al. 2000). The cluster also occupies a different orientation as a function of the presence of various inhibitors as is seen with MOA-stilbene and stigmatellin. A similar result has also been seen with the b 6 f complex in the presence of copper (Roberts et al. 2002) or DB- MIB (Schoepp et al. 1999) using oriented membrane samples. Second, a protease digestion assay indicated that in R. capsulatus bc 1 complex, the degree of proteolysis of the Fe S subunit linker region decreased and increased following the addition of stigmatellin and myxothiazol, respectively (Valkova-Valchanova et al. 2000). In the case of the b 6 f complex it has also been found that increasing the viscosity of the medium decreased the rate of cyt f turnover (Heimann et al. 2000). Third, steered molecular dynamics studies suggested that the movement was possibly guided by hydrogen bond breakage and formation, and predicted a displacement of about 2 Å of the ef loop of cyt b (Izrailev et al. 1999). In addition, a wealth of important information on the movement of the Fe S subunit was obtained by studying various bc 1 complex mutants of species that are amenable to genetic analyses as described below. As the membrane anchor of the Fe S subunit stays fixed in the various structures, the rotation of the cluster domain requires conformational changes of the flexible region linking these two domains that acts as a hinge (Figure 2). Results have shown that in bacterial species like Rhodobacter or in yeast, when this linker was rendered too rigid (by creation of an intra subunit disulfide bridge or by replacement of appropriate amino acids with proline residues) (Tian et al. 1998, 1999; Darrouzet et al. 2000a) or too long (by insertions of extra alanine residues) (Darrouzet et al. 2000b; Nett et al. 2000; Obungu et al. 2000), the function of the bc 1 complex was impaired to varying extents in terms of their steady-state or single turnover activity, inhibitor sensitivity, K m for substrate and E m of the [2Fe2S] cluster. Similar decreases in enzyme activity have also been seen when inter-subunit disulfide bridges that would impede the movement of the Fe S subunit were engineered (Xiao et al. 2000). Upon addition of reducing agents, the activity of the bc 1 complex could be restored at least partially by the breaking of disulfide bridges (Tian et al. 1999; Xiao et al. 2000). In contrast, shortening the hinge region had little effect on the activity of the enzyme, although it often affected drastically the assembly of the Fe S subunit into the bc 1 complex (Darrouzet et al. 2000a). Among the various mutants, those with an insertion of alanine residues in R. capsulatus (+nala