Structural Basis of Multifunctional Bovine Mitochondrial Cytochrome bc 1 Complex

The mitochondrial cytochrome bc ₁ complex is a multifunctional membrane protein complex. Itcatalyzes electron transfer, proton translocation, peptide processing, and superoxide generation.Crystal structure data at 2.9 Å resolution not only establishes the location of the redox centersand inhibitor binding sites, but also suggests a movement of the head domain of the iron–sulfurprotein (ISP) during bc ₁ catalysis and inhibition of peptide-processing activity during complexmaturation. The functional importance of the movement of extramembrane (head) domain ofISP in the bc ₁ complex is confirmed by analysis of the Rhodobacter sphaeroides bc ₁ complexmutants with increased rigidity in the ISP neck and by the determination of rate constants foracid/base-induced intramolecular electron transfer between [2Fe–2S] and heme c ₁ in nativeand inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovineheart mitochondrial bc ₁ complex by nonionic detergent at concentrations that inactivate electrontransfer activity. This peptide-processing activity is shown to be associated with subunits Iand II by cloning, overexpression and in vitro reconstitution. The superoxide-generation siteof the cytochrome bc ₁ complex is located at reduced b _L and Q^–. The reaction is membranepotential-, and cytochrome c-dependent.     

Evidence for the head domain movement of the rieske iron-sulfur protein in electron transfer reaction of the cytochrome bc1 complex

The three-dimensional structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane domain (head) of the Rieske iron-sulfur protein (ISP) may play an important role in electron transfer. Such movement requires flexibility in the neck region of ISP, since the head and transmembrane domains of the protein are rather rigid. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with cysteine substitution at various positions in the ISP neck (residues 39-48) were generated and characterized. The mutants with a single cysteine substitution at Ala42 or Val44 and a double cysteine substitution at Val44 and Ala46 (VQA-CQC) or at Ala42 and Ala46 (ADVQA-CDVQC) have photosynthetic growth rates comparable with that of complement cells. Chromatophore membrane and intracytoplasmic membrane (ICM) prepared from these mutants have cytochrome bc1 complex activity similar to that in the complement membranes, indicating that flexibility of the neck region of ISP was not affected by these cysteine substitutions. Mutants with a double cysteine substitution at Ala42 and Val44 (ADV-CDC) or at Pro40 and Ala42 (PSA-CSC) have a retarded (50%) or no photosynthetic growth rate, respectively. The ADV-CDC or PSA-CSC mutant ICM contains 20 or 0% of the cytochrome bc1 complex activity found in the complement ICM. However, activity can be restored by the treatment with beta-mercaptoethanol (beta-ME). The restored activity is diminished upon removal of beta-ME but is retained if the beta-ME-treated membrane is treated with the sulfhydryl reagent N-ethylmaleimide or p-chloromercuribenzoic acid. These results indicate that the loss of bc1 complex activity in the ADV-CDC or PSA-CSC mutant membranes is due to disulfide bond formation, which increases the rigidity of ISP neck and, in turn, decreases the mobility of the head domain. Using the conditions developed for the isolation of His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) is obtained from all of the double cysteine-substituted mutants. This suggests that introduction of two cysteines in the neck region of ISP weakens the interactions between cytochromes b, ISP, and subunit IV.     

Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein

To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc(1) complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc(1) activity. Reduction of this disulfide bond by beta-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc(1) complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c(1).     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

pH-induced intramolecular electron transfer between the iron-sulfur protein and cytochrome c(1) in bovine cytochrome bc(1) complex

Structural analysis of the bc(1) complex suggests that the extra membrane domain of iron-sulfur protein (ISP) undergoes substantial movement during the catalytic cycle. Binding of Qo site inhibitors to this complex affects the mobility of ISP. Taking advantage of the difference in the pH dependence of the redox midpoint potentials of cytochrome c(1) and ISP, we have measured electron transfer between the [2Fe-2S] cluster and heme c(1) in native and inhibitor-treated partially reduced cytochrome bc(1) complexes. The rate of the pH-induced cytochrome c(1) reduction can be estimated by conventional stopped-flow techniques (t1/2, 1-2 ms), whereas the rate of cytochrome c(1) oxidation is too high for stopped-flow measurement. These results suggest that oxidized ISP has a higher mobility than reduced ISP and that the movement of reduced ISP may require an energy input from another component. In the 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT)-inhibited complex, the rate of cytochrome c(1) reduction is greatly decreased to a t1/2 of approximately 2.8 s. An even lower rate is observed with the stigmatellin-treated complex. These results support the idea that UHDBT and stigmatellin arrest the [2Fe-2S] cluster at a fixed position, 31 A from heme c(1), making electron transfer very slow.     

A novel electron transfer mechanism suggested by crystallographic studies of mitochondrial cytochrome bc1 complex.

The crystal structure of bovine mitochondrial cytochrome bc1 complex, an integral membrane protein complex of 11 different subunits with a total molecular mass of 242 kDa, demonstrated a tightly associated dimer consisting of three major regions: a matrix region primarily made of subunits core1, core2, 6, and 9; a transmembrane-helix region of 26 helices in the dimer contributed by cytochrome b, cytochrome c1, the Rieske iron-sulfur protein (ISP), subunits 7, 10, and 11; and an intermembrane-space region composed of extramembrane domains of ISP, cytochrome c1, and subunit 8. The structure also revealed the positions of and distances between irons of prosthetic groups, and two symmetry related cavities in the transmembrane-helix region upon dimerization of the bc1 complex. Extensive crystallographic studies on crystals of bc1 complexed with inhibitors of electron transfer identified binding pockets for both Qo and Qi site inhibitors. Discrete binding sites for subtypes of Qo site inhibitors have been mapped onto the Qo binding pocket, and bindings of different subtypes of Qo site inhibitors are capable of inducing dramatic conformational changes in the extramembrane domain of ISP. A novel electron transfer mechanism for the bc1 complex consistent with crystallographic observations is discussed.     

The crystal structure of mitochondrial cytochrome bc1 in complex with famoxadone: the role of aromatic-aromatic interaction in inhibition

Ubiquinol cytochrome c oxido-reductase (EC. 1.10.2.2, bc1) is an integral membrane protein complex essential to cellular respiration. Structures of the 11-subunit mitochondrial bc1 complex were determined with and without the fungicide famoxadone. Specific inhibition by famoxadone is achieved through a coordinated optimization of aromatic-aromatic interactions where conformational rearrangements in famoxadone and in residues lining the inhibitor-binding pocket produce a network of aromatic-aromatic interactions that mimic the crystal lattice of benzene. The profound aromatic-aromatic interactions as supported by prior mutagenesis provide a structural basis for specific protein-ligand interaction in a hydrophobic environment. Dramatic conformational changes, both in cyt. b and ISP subunits in the inhibitor-protein complex, confer experimental evidence for a functional role of cytochrome b in the induced conformational arrest of ISP and allow the identification of a possible intrasubunit signal transduction pathway that controls the movement of ISP. These results support an inhibitory mechanism that is consistent with the requirement for ISP movement in the electron transfer of this complex.     

Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex.

The cytochrome bc1 complex from bovine heart mitochondria is a multi-functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron-sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 A between bL heme and bH heme and of 27 A between bL heme and the iron-sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 A between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co-crystallized with the Qu site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.     

Steered molecular dynamics simulation of the Rieske subunit motion in the cytochrome bc(1) complex.

Crystallographic structures of the mitochondrial ubiquinol/cytochrome c oxidoreductase (cytochrome bc(1) complex) suggest that the mechanism of quinol oxidation by the bc(1) complex involves a substantial movement of the soluble head of the Rieske iron-sulfur protein (ISP) between reaction domains in cytochrome b and cytochrome c(1) subunits. In this paper we report the results of steered molecular dynamics simulations inducing, through an applied torque within 1 ns, a 56 degrees rotation of the soluble domain of ISP. For this purpose, a solvated structure of the bc(1) complex in a phospholipid bilayer (a total of 206,720 atoms) was constructed. A subset of 91,061 atoms was actually simulated with 45,131 moving atoms. Point charge distributions for the force field parametrization of heme groups and the Fe(2)S(2) cluster of the Rieske protein included in the simulated complex were determined. The simulations showed that rotation of the soluble domain of ISP is actually feasible. Several metastable conformations of the ISP during its rotation were identified and the interactions stabilizing the initial, final, and intermediate positions of the soluble head of the ISP domain were characterized. A pathway for proton conduction from the Q(o) site to the solvent via a water channel has been identified.     

Mitochondrial superoxide radical formation is controlled by electron bifurcation to the high and low potential pathways

The generation of oxygen radicals in biological systems and their sites of intracellular release have been subject of numerous studies in the last decades. Based on these studies mitochondria are considered to be the major source of intracellular oxygen radicals. Although this finding is more or less accepted, the mechanism of univalent oxygen reduction in mitochondria is still obscure. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation at the cytochrome bc 1 complex. Recent studies with genetically mutated mitochondria have made it clear that electron bifurcation from ubiquinol to the cytochrome bc 1 complex requires the free mobility of the head domain of the Rieske iron-sulfur protein. On the other hand, it has been long known that inhibition of electron bifurcation by antimycin A causes leakage of single electrons to dioxygen, which results in the release of superoxide radicals. These findings lead us to study whether hindrance of the interaction of ubiquinol with the cytochrome bc 1 complex is the regulator of single electron diversion to oxygen. Hindrance of electron bifurcation was observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc 1 complex is inserted. Irrespective of whether the fluidity of the membrane lipids was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting an effect on the mobility of the head domain of the Rieske iron-sulfur protein. This revealed the involvement of the ubiquinol cytochrome bc 1 redox couple in mitochondrial superoxide formation. The regulator, which controls leakage of electrons to oxygen, appears to be the electron-branching activity of the cytochrome bc 1 complex.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.     

Mitochondrial Superoxide Radical Formation is Controlled by Electron Bifurcation to the High and Low Potential Pathways

The generation of oxygen radicals in biological systems and their sites of intracellular release have been subject of numerous studies in the last decades. Based on these studies mitochondria are considered to be the major source of intracellular oxygen radicals. Although this finding is more or less accepted, the mechanism of univalent oxygen reduction in mitochondria is still obscure. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation at the cytochrome bc 1 complex. Recent studies with genetically mutated mitochondria have made it clear that electron bifurcation from ubiquinol to the cytochrome bc 1 complex requires the free mobility of the head domain of the Rieske iron-sulfur protein. On the other hand, it has been long known that inhibition of electron bifurcation by antimycin A causes leakage of single electrons to dioxygen, which results in the release of superoxide radicals. These findings lead us to study whether hindrance of the interaction of ubiquinol with the cytochrome bc 1 complex is the regulator of single electron diversion to oxygen. Hindrance of electron bifurcation was observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc 1 complex is inserted. Irrespective of whether the fluidity of the membrane lipids was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting an effect on the mobility of the head domain of the Rieske iron-sulfur protein. This revealed the involvement of the ubiquinol cytochrome bc 1 redox couple in mitochondrial superoxide formation. The regulator, which controls leakage of electrons to oxygen, appears to be the electron-branching activity of the cytochrome bc 1 complex.     

The modified Q-cycle explains the apparent mismatch between the kinetics of reduction of cytochromes c1 and bH in the bc1 complex

Crystallographic structures of the bc1 complex from different sources have provided evidence that a movement of the Rieske iron-sulfur protein (ISP) extrinsic domain is essential for catalysis. This dynamic feature has opened up the question of what limits electron transfer, and several authors have suggested that movement of the ISP head, or gating of such movement, is rate-limiting. Measurements of the kinetics of cytochromes and of the electrochromic shift of carotenoids, following flash activation through the reaction center in chromatophore membranes from Rhodobacter sphaeroides, have allowed us to demonstrate that: (i) ubiquinol oxidation at the Qo-site of the bc1 complex has the same rate in the absence or presence of antimycin bound at the Qi-site, and is the reaction limiting turnover. (ii) Activation energies for transient processes to which movement of the ISP must contribute are much lower than that of the rate-limiting step. (iii) Comparison of experimental data with a simple mathematical model demonstrates that the kinetics of reduction of cytochromes c1 and bH are fully explained by the modified Q-cycle. (iv) All rates for processes associated with movement of the ISP are more rapid by at least an order of magnitude than the rate of ubiquinol oxidation. (v) Movement of the ISP head does not introduce a significant delay in reduction of the high potential chain by quinol, and it is not necessary to invoke such a delay to explain the kinetic disparity between the kinetics of reduction of cytochromes c1 and bH.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Movement of the Rieske iron-sulfur protein in the p-side bulk aqueous phase: effect of lumenal viscosity on redox reactions of the cytochrome b6f complex

Based on the atomic structures of the mitochondrial cytochrome bc(1) complex, it has been proposed that the soluble domain of the [2Fe-2S] Rieske iron-sulfur protein (ISP) must rotate by ca. 60 degrees and translate through an appreciable distance between two binding sites, proximal to cytochrome c(1) and to the lumen-side quinol binding site. Such motional freedom implies that the electron-transfer rate should be affected by the lumenal viscosity. The flash-induced oxidation of cytochrome f, the chloroplast analogue of cytochrome c(1), was found to be inhibited reversibly by increased lumenal viscosity, as was the subsequent reduction of both cytochrome b(6) and cytochrome f. The rates of these three redox reactions correlated inversely with lumenal viscosity over a viscosity range of 1-10 cP. Reduction of cytochrome b(6) and cytochrome f was not concerted. The rate of cytochrome f reduction was observed to be approximately half that of cytochrome b(6) regardless of the actual viscosity, implying that the path length traversed by the ISP in reduction of cytochrome f is twice that of cytochrome b(6). This suggests that upon initiation of electron transfer by a light flash, cytochrome b(6) reduction requires movement of reduced ISP from an initial position predominantly proximal to cytochrome f, apparently favored by the reduced ISP, to the quinol binding site at which the oxidant-induced reduction of cytochrome b(6) is initiated. Subsequent reduction of cytochrome f requires the additional movement of the ISP back to a site proximal to cytochromef. There is no discernible viscosity dependence for cytochrome b(6) reduction under oxidizing conditions, presumably because the oxidized ISP preferentially binds proximal to the quinone binding niche. The dependence of the cytochrome redox reaction on ambient viscosity implies that the tethered diffusional motion of the ISP is part of the rate limitation for charge transfer through the b(6)f complex.     

Evidence for the intertwined dimer of the cytochrome bc(1) complex in solution

To confirm that the cytochrome bc(1) complex exists as a dimer with intertwining Rieske iron-sulfur proteins in solution, four Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes containing two pairs of cysteine substitutions, one in the interface between the head domain of iron-sulfur protein (ISP) and cytochrome b and the other between the tail domain of ISP and cytochrome b, were generated and characterized. They are: K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb), K70C(ISP)/A185C(cytb).P33C(ISP)/M92C (cytb), K70C (ISP)/A185C(cytb).L34C(ISP)/V64C(cytb), and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb). The K70C(ISP)/A185C(cytb) cysteine pair cross-links the head domain of ISP and cytochrome b; the P33C(ISP)/G89C(cytb), P33C(ISP)/M92C (cytb), L34C(ISP)/V64C(cytb), and N36C(ISP)/G89C(cytb) cysteine pairs cross-link the tail domain of ISP and cytochrome b. An adduct protein with an apparent molecular mass of 128 kDa containing two cytochrome b and two ISP proteins is detected in the K70C(ISP)/A185C(cytb).P33C(ISP)/G89C(cytb) and K70C(ISP)/A185C(cytb).N36C(ISP)/G89C(cytb) mutant complexes, confirming that the bc(1) complex exists as a dimer with intertwining ISPs. The loss of activity in these two double-cysteine-pair mutant complexes was attributed to the disulfide bond between the head domain of ISP and cytochrome b and not the one between the tail domain of ISP and cytochrome b.     

Structural basis for the mechanism of electron bifurcation at the quinol oxidation site of the cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> complex

At the heart of the Q cycle hypothesis, the cytochrome bc1 complex (bc1) is required to separate the two electrons from a quinol molecule at the quinol oxidation site. Recent studies have brought to light an intricate mechanism for this bifurcated electron transfer. A survey of the protein data bank shows 30 entries for the structures of bc1 and the homologous b6 f complex. These structures provide considerable insights into the structural organization of mitochondrial, bacterial, and plant enzymes. Crystallographic binding studies of bc1 with either quinone reduction (QN) and/or quinol oxidation (QP) site inhibitors offer atomic details on how these compounds interact with residues at their respective sites. Most importantly, the different locations and apparent flexibility observed in crystals for the extrinsic domain of the iron-sulfur protein (ISP) subunit suggest a mechanism for electron bifurcation at the QP site. Analyses of various inhibitor-bound structures revealed two classes of QP site inhibitors: Pm inhibitors that promote ISP mobility and Pf inhibitors that favor the fixation of the ISP conformation. Those analyses also shed light on a possible process by which the ISP motion switch is controlled. The first phase reduction of ISP is shown to be comparable to the reduction of the bL heme by pre-steady state kinetic analysis, whereas the second phase reduction of ISP share similar kinetics with the reduction of the bH heme. The reduction of cyt c1 is measured much slower, indicating that the reduced ISP remains bound at the QP site until the reduced heme bL is oxidized by the heme bH and supporting the existence of a control mechanism for the ISP motion switch.     

X-ray structure of the dimeric cytochrome bc(1) complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution

The respiratory cytochrome bc(1) complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c(1) changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-? resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c(1) shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c(1) surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c(1) and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c(552), the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding.     

The ubiquinol/bc1 redox couple regulates mitochondrial oxygen radical formation

The generation of oxygen radicals in biological systems and their sites of intracellular release were subject of numerous studies in the last decades. Based on these studies mitochondria were considered as the major source of intracellular oxygen radicals. Although this finding is more or less accepted the mechanism of univalent oxygen reduction in mitochondria is still obscure. One of the most critical electron transfer steps of the respiratory chain is the electron bifurcation at the bc1 complex. From recent studies with genetically mutated mitochondria it became clear that electron bifurcation from ubiquinol to the bc1 complex requires an underanged mobility of the head domain of the Rieske iron sulfur protein. On the other hand it is long known that inhibition of electron bifurcation by antimycin A causes the leakage of single electrons to dioxygen, which results in the release of O2*- radicals. These findings made us to prove whether the impediment of the interaction of ubiquinol with the bc1 complex is the regulator of single electron diversion to oxygen. Impediment of electron bifurcation was observed following alterations of the physical state of membrane phospholipids in which the bc1 complex is inserted. Irrespectively, whether the fluidity of membrane lipids was elevated or decreased electron flow rates to the Rieske iron sulfur protein and to low potential cytochrome b were drastically reduced. Concomitantly O2*- radicals were released from these mitochondria, suggesting an effect on the mobility of the head domain of the Rieske iron sulfur protein. These results including the well known effect of antimycin A revealed the involvement of the ubiquinol bc1 redox couple in mitochondrial O2*- formation. The regulator which controls leakage of electrons to oxygen appears to be the electron branching activity of the bc1 complex.