Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.     

Cytochrome bc1 complexes of microorganisms.

The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae.     

Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans

A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron-sulfur protein on the basis of its molecular weight and the presence of an EPR-detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle.     

The Rhodospirillum rubrum cytochrome bc1 complex: redox properties, inhibitor sensitivity and proton pumping.

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.     

DCCD binds to cytochrome b6 of a cytochrome bf complex isolated from spinach chloroplasts and inhibits proton translocation.

Radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD) was bound selectively in a time- and concentration-dependent manner to cytochrome b6 of an enzymatically active cytochrome bf complex isolated from spinach chloroplasts. Maximum labeling of cytochrome b6 was observed with 30 nmol DCCD per nmol cytochrome b6 in the cytochrome bf complex incubated for 30-60 min at 12 degrees C. After incubation of the cytochrome bf complex with DCCD under these conditions, the rate of proton ejection in the complex reconstituted into liposomes was decreased approximately 65-70% when compared to controls; however, under these same conditions the rate of electron transfer through either the soluble bf complex or the complex reconstituted into liposomes was only decreased around 20%. These results suggest that the mechanism of proton translocation through the cytochrome bf complex of spinach chloroplasts is similar to that of the cytochrome bc1 complex from yeast mitochondria in which proton pumping but not electron transfer is also inhibited by DCCD (D. S. Beattie and A. Villalobo, 1982, J. Biol. Chem. 257, 14,745-14,752).     

DCCD inhibits the reactions of the iron-sulfur protein in Rhodobacter sphaeroides chromatophores

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).     

Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes

An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain.     

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

Spheroplasts from aerobically grown wild-type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc1 complex by myxothiazol. Coupled to this activity, which involves only b-type cytochromes, there is translocation of 1.5-1.9 h+/e- across the cytoplasmic membrane. Similar H+ translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa3 to which it is structurally related.     

Chemical modification of the mitochondrial bc1 complex by N,N'-dicyclohexylcarbodiimide inhibits proton translocation

We report here that N,N'-dicyclohexylcarbodiimide (DCCD) decreases the H/2e stoichiometry of the cytochrome bc1 complex from 3.8 +/- 0.2 (10) to 2.1 +/- 0.1 (8) but has only a minimal effect on the H/2e ratio of cytochrome oxidase under the relatively mild conditions used. The effect on the bc1 complex cannot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage. We conclude that DCCD is an inhibitor of proton translocation within the bc1 complex. There are three possible explanations of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reactions but not electron transport, (c) DCCD could prevent the conduction of the translocated proton to the external phase.     

Identification of a stable ubisemiquinone and characterization of the effects of ubiquinone oxidation-reduction status on the Rieske iron-sulfur protein in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans

The ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans exhibits a thermodynamically stable ubisemiquinone radical detectable by EPR spectroscopy. The radical is centered at g = 2.004, is sensitive to antimycin, and has a midpoint potential at pH 8.5 of +42 mV. These properties are very similar to those of the stable ubisemiquinone (Qi) previously characterized in the cytochrome bc1 complexes of mitochondria. The micro-environment of the Rieske iron-sulfur cluster in the Paracoccus cytochrome bc1 complex changes in parallel with the redox state of the ubiquinone pool. This change is manifested as shifts in the gx, gy, and gz values of the iron-sulfur cluster EPR signal from 1.80, 1.89, and 2.02 to 1.76, 1.90, and 2.03, respectively, as ubiquinone is reduced to ubiquinol. The spectral shift is accompanied by a broadening of the signal and follows a two electron reduction curve, with a midpoint potential at pH 8.5 of +30 mV. A hydroxy analogue of ubiquinone, UHDBT, which inhibits respiration in the cytochrome bc1 complex, shifts the gx, gy, and gz values of the iron-sulfur cluster EPR signal to 1.78, 1.89, and 2.03, respectively, and raises the midpoint potential of the iron-sulfur cluster at pH 7.5 from +265 to +320 mV. These changes in the micro-environment of the Paracoccus Rieske iron-sulfur cluster are like those elicited in mitochondria. These results indicate that the cytochrome bc1 complex of P. denitrificans has a binding site for ubisemiquinone and that this site confers properties on the bound ubisemiquinone similar to those in mitochondria. In addition, the line shape of the Rieske iron-sulfur cluster changes in response to the oxidation-reduction status of ubiquinone, and the midpoint of the iron-sulfur cluster increases in the presence of a hydroxyquinone analogue of ubiquinone. The latter results are also similar to those observed in the mitochondrial cytochrome bc1 complex. However, unlike the mitochondrial complexes, which contain eight to 11 polypeptides and are thought to contain distinct quinone binding proteins, the Paracoccus cytochrome bc1 complex contains only three polypeptide subunits, cytochromes b, c1, and iron-sulfur protein. The ubisemiquinone binding site and the site at which ubiquinone and/or ubiquinol bind to affect the Rieske iron-sulfur cluster in Paracoccus thus exist in the absence of any distinct quinone binding proteins and must be composed of domains contributed by the cytochromes and/or iron-sulfur protein.     

Insights from the structure of the yeast cytochrome bc1 complex: crystallization of membrane proteins with antibody fragments

The ubiquinol:cytochrome c oxidoreductase (EC 1.20.2.2, QCR or cytochrome bc1 complex) is a component of respiratory and photosynthetic electron transfer chains in mitochondria and bacteria. The complex transfers electrons from quinol to cytochrome c. Electron transfer is coupled to proton translocation across the lipid bilayer, thereby generating an electrochemical proton gradient, which conserves the free energy of the redox reaction. The yeast complex was crystallized with antibody Fv fragments, a promising technique to obtain well-ordered crystals from membrane proteins. The high-resolution structure of the yeast protein reveals details of the catalytic sites of the complex, which are important for electron and proton transfer.     

Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.     

Inhibitor-complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides

The cytochrome bc(1) complex (bc(1)) is a major contributor to the proton motive force across the membrane by coupling electron transfer to proton translocation. The crystal structures of wild type and mutant bc(1) complexes from the photosynthetic purple bacterium Rhodobacter sphaeroides (Rsbc(1)), stabilized with the quinol oxidation (Q(P)) site inhibitor stigmatellin alone or in combination with the quinone reduction (Q(N)) site inhibitor antimycin, were determined. The high quality electron density permitted assignments of a new metal-binding site to the cytochrome c(1) subunit and a number of lipid and detergent molecules. Structural differences between Rsbc(1) and its mitochondrial counterparts are mostly extra membranous and provide a basis for understanding the function of the predominantly longer sequences in the bacterial subunits. Functional implications for the bc(1) complex are derived from analyses of 10 independent molecules in various crystal forms and from comparisons with mitochondrial complexes.     

Inhibition of reversed electron transfer and proton transport in the beef heart cytochrome bc1 complex by chemical modification

Chemical modification of the bovine heart cytochrome bc1 complex with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) has been reported to inhibit the proton pumping activity without affecting the rate of electron transfer to ferricytochrome c. This study aims to examine the effect of EEDQ on energy-linked reversed electron transfer in the bc1 complex reconstituted into potassium-loaded phospholipid vesicles. Generation of a valinomycin-mediated potassium-diffusion potential induced the reduction of cytochrome b in the reconstituted bc1 complex in the presence of sodium ascorbate. The time course of the cytochrome b reduction was well correlated with that of the absorbance change of safranine, an optical probe for measuring membrane potential. Treatment of the bc1 complex with EEDQ caused a decrease in the potential-induced reduction of cytochrome b as well as in the proton translocation activity. But a significant loss in the ubiquinol-cytochrome c reducing activity was not observed in the EEDQ-treated bc1 complex. The time- and concentration-dependent effect of EEDQ on the reversed electron transfer was well correlated with that of the proton translocation activity of the bc1 complex. These findings strongly support the idea that the potential-induced reversal of electron transfer is coupled to the reverse flow of protons in the cytochrome bc1 complex.     

Aspartate-187 of cytochrome b is not needed for DCCD inhibition of ubiquinol: cytochrome c oxidoreductase in Rhodobacter sphaeroides chromatophores

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD.     

The role of extra fragment at the C-terminal of cytochrome b (Residues 421-445) in the cytochrome bc1 complex from Rhodobacter sphaeroides

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.     

Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment

BACKGROUND: The cytochrome bc(1) complex is part of the energy conversion machinery of the respiratory and photosynthetic electron transfer chains. This integral membrane protein complex catalyzes electron transfer from ubiquinol to cytochrome c. It couples the electron transfer to the electrogenic translocation of protons across the membrane via a so-called Q cycle mechanism. RESULTS: The cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae was crystallized together with a bound antibody Fv fragment. The structure was determined at 2.3 A resolution using multiple isomorphous replacement, and refined to a crystallographic R factor of 22.2% (R(free) = 25.4%). The complex is present as a homodimer. Each 'monomer' of the refined model includes 2178 amino acid residues of subunits COR1, QCR2, COB, CYT1, RIP1, QCR6, QCR7, QCR8 and QCR9 of the cytochrome bc(1) complex and of the polypeptides V(H) and V(L) of the Fv fragment, the cofactors heme b(H), heme b(L), heme c(1), the [2Fe-2S] cluster and 346 water molecules. The Fv fragment binds to the extrinsic domain of the [2Fe-2S] Rieske protein and is essential for formation of the crystal lattice. CONCLUSIONS: The approach to crystallize membrane proteins as complexes with specific antibody fragments appears to be of general importance. The structure of the yeast cytochrome bc(1) complex reveals in detail the binding sites of the natural substrate coenzyme Q6 and the inhibitor stigmatellin. Buried water molecules close to the binding sites suggest possible pathways for proton uptake and release. A comparison with other cytochrome bc(1) complexes shows features that are specific to yeast.     

Similar transition states mediate the Q-cycle and superoxide production by the cytochrome bc1 complex

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q(o)) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q(o) site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q(o) site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O(2) reduction; 2) the Q(o) site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q(o) site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.     

Role of PCDH10 and its hypermethylation in human gastric cancer

Cytochrome bc(1) complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c(2)) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc(1) complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c(1) with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c(1)-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5? resolution.     

Carboxyl residues in the iron-sulfur protein are involved in the proton pumping activity of P. denitrificans bc(1) complex.

A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the complex. Direct measurement of the binding of EEDQ to the complex subunits, performed in the presence of the fluorescent hydrophobic nucleophile 4'-[(aminoacetamido)methyl]fluorescein (AMF), showed that the iron-sulfur protein (ISP) and cytochrome c(1) were labeled by EEDQ, whereas cytochrome b was not. Tryptic digestion and sequencing analysis of the fluorescent fragment of the ISP revealed this to consist of a segment with six acidic residues, among which the highly conserved aspartate 160 is present. Analogous experiments on DCCD binding showed that all the three subunits of the complex were labeled. However, DCCD concentration dependence of carboxyl residue modification in the individual subunits and of proton pumping activity showed that the decrease of the H(+)/e(-) ratio correlated only with the modification of the ISP. Tryptic digestion of labeled ISP and sequencing analysis of the fluorescent fragment gave results superimposable upon those obtained with EEDQ. Chymotryptic digestion and sequencing analysis of the single fluorescent fragment of cytochrome b showed that this fragment contained glutamate 174 and aspartate 187. We conclude that, in the P. denitrificans bc(1) complex, carboxyl residues in cytochrome b do not appear to be critically involved in the proton pump mechanism of the complex.