Cytochrome bc1 complex [2Fe-2S] cluster and its interaction with ubiquinone and ubihydroquinone at the Qo site: a double-occupancy Qo site model.

The ubiquinone complement of Rhodobacter capsulatus chromatophore membranes has been characterized by its isooctane solvent extractability and electrochemistry; we find that the main ubiquinone pool (Qpool) amounts to about 80% of the total ubiquinone and has an Em7 value close to 90 mV. To investigate the interactions of ubiquinone with the cyt bc1 complex, we have examined the distinctive EPR line shapes of the [2Fe-2S] cluster of the cyt bc1 complex when the Qpool-cyt bc1 complex interactions are modulated by changing the numbers of Q or QH2 present (by solvent extraction and reconstitution), by the exposure of the [2Fe-2S] to the Qpool in different redox states, by the presence of inhibitors specific for the Qo site (myxothiazol and stigmatellin) and Qi site (antimycin), and by site-specific mutations of side chains of the cyt b polypeptide (mutants F144L and F144G) previously identified as important for Qo site structure. Evidence suggests that the Qo site can accommodate two ubiquinone molecules. One (designated Qos) is bound relatively strongly and is second only to the ubiquinone of the QA site of the reaction center in its resistance to solvent extraction. In this strong interaction, the Qo site binds Q and QH2 with approximately equal affinities. Their bound states are distinguished by their effects on the [2Fe-2S] cluster spectral feature at gx at 1.783 (Q) and gx at 1.777 (QH2); titration of the line-shape change reveals an Em7 value of approximately 95 mV. The other molecule (Qow) is bound more weakly, in the same range as the ubiquinone of the QB site of the reaction center. Again, the affinities of the Q form (gx at 1.800) and QH2 form (gx at 1.777) are nearly equal, and the Em7 value measured is approximately 80 mV. These results are discussed in terms of earlier EPR analyses of the cyt bc1 complexes of other systems. A Qo site double-occupancy model is considered that builds on the previous model based on Qo site mutants [Robertson, D. E., Daldal, F.,& Dutton, P. L. (1990) Biochemistry 29, 11249-11260] and includes the recent suggestion that two of the [2F3-2S] cluster ligands of the R. capsulatus cyt bc1 complex are histidines [Gurbiel, R. J. Ohnishi, T., Robertson, D. E. Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. We speculate that the cyt bc1 complex complexes a full enzymatic turnover without necessary exchange of ubiquinone with the Qpool.     

Protein-protein interactions between cytochrome b and the Fe-S protein subunits during QH2 oxidation and large-scale domain movement in the bc1 complex

The ubihydroquinone:cytochrome (cyt) c oxidoreductase, or bc(1) complex, and its homologue the b(6)f complex are key components of respiratory and photosynthetic electron transport chains as they contribute to the generation of an electrochemical gradient used by the ATP synthase to produce ATP. The bc(1) complex has two catalytic domains, ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) sites, that are located on each side of the membrane. The key to the energetic efficiency of this enzyme relies upon the occurrence of a unique electron bifurcation reaction at its Q(o) site. Recently, several lines of evidence have converged to establish that in the bc(1) complex the extrinsic domain of the Fe-S subunit that contains a [2Fe2S] metal cluster moves during catalysis to shuttle electrons between the Q(o) site and c(1) heme. While this step is required for electron bifurcation, available data also suggest that the movement might be controlled to ensure maximal energetic efficiency [Darrouzet et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4567-4572]. To gain insight into the plausible control mechanism, we used a biochemical genetic approach to define the different regions of the bc(1) complex that might interact with each other. Previously, we found that a mutation located at position L286 of the ef loop of Rhodobacter capsulatus cyt b could alleviate movement impairment resulting from a mutation in the hinge region, linking the [2Fe2S] cluster domain to the membrane anchor of the Fe-S subunit. Here we report that various substitutions at position 288 on the opposite side of the ef loop also impair Q(o) site catalysis. In particular, we note that while most of the substitutions affect only QH(2) oxidation, yet others like T288S also hinder the rate of the movement of the Fe-S subunit. Thus, position 288 of cyt b appears to be important for both the QH(2) oxidation and the movement of the Fe-S subunit. Moreover, we found that, upon substitution of T288 by other amino acids, additional compensatory mutations located at the [2Fe2S] cluster or the hinge domains of the Fe-S subunit, or on the cd loop of cyt b, arise readily to alleviate these defects. These studies indicate that intimate protein-protein interactions occur between cyt b and the Fe-S subunits to sustain fast movement and efficient QH(2) oxidation and highlight the critical dual role the ef loop of cyt b to fine-tune the docking and movement of the Fe-S subunit during Q(o) site catalysis.     

The [2Fe-2S] cluster E(m) as an indicator of the iron-sulfur subunit position in the ubihydroquinone oxidation site of the cytochrome bc1 complex.

Recent crystallographic and kinetic data have revealed the crucial role of the large scale domain movement of the iron-sulfur subunit [2Fe-2S] cluster domain during the ubihydroquinone oxidation reaction catalyzed by the cytochrome bc(1) complex. Previously, the electron paramagnetic resonance signature of the [2Fe-2S] cluster and its redox midpoint potential (E(m)) value have been used extensively to characterize the interactions of the [2Fe-2S] cluster with the occupants of the ubihydroquinone oxidation (Q(o)) catalytic site. In this work we analyze these interactions in various iron-sulfur subunit mutants that carry mutations in its flexible hinge region. We show that the E(m) increases of the iron-sulfur subunit [2Fe-2S] cluster induced either by these mutations or by the addition of stigmatellin do not act synergistically. Moreover, the E(m) increases disappear in the presence of class I inhibitors like myxothiazol. Because various inhibitors are known to affect the location of the iron-sulfur subunit cluster domain, the measured E(m) value of the [2Fe-2S] cluster therefore reflects its equilibrium position in the Q(o) site. We also demonstrate the existence in this site of a location where the E(m) of the cluster is increased by about 150 mV and discuss its possible implications in term of Q(o) site catalysis and energetics.     

Primary steps in the energy conversion reaction of the cytochrome bc1 complex Qo site

The primary energy conversion (Qo) site of the cytochrome bc1 complex is flanked by both high- and low-potential redox cofactors, the [2Fe-2S] cluster and cytochrome bL, respectively. From the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectral g(x)-band and line shape to the degree and type of Qo site occupants, we have proposed a double-occupancy model for the Qo site by ubiquinone in Rhodobacter capsulatus membrane vesicles containing the cytochrome bc1 complex. Biophysical and biochemical experiments have confirmed the double occupancy model and from a combination of these results and the available cytochrome bc1 crystal structures we suggest that the two ubiquinone molecules in the Qo site serve distinct catalytic roles. We propose that the strongly bound ubiquinone, termed Qos, is close to the [2Fe-2S] cluster, where it remains tightly associated with the Qo site during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Qow, is distal to the [2Fe-2S] cluster and can exchange with the membrane Qpool on a time scale much faster than the turnover, acting as the substrate. The crystallographic data demonstrates that the FeS subunit can adopt different positions. Our own observations show that the equilibrium position of the reduced FeS subunit is proximal to the Qo site. On the basis of this, we also report preliminary results modeling the electron transfer reactions that can occur in the cytochrome bc1 complex and show that because of the strong distance dependence of electron transfer, significant movement of the FeS subunit must occur in order for the complex to be able to turn over at the experimental observed rates.     

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.     

The inhibitor DBMIB provides insight into the functional architecture of the Qo site in the cytochrome b6f complex

Previously [Roberts, A. G., and Kramer, D. M. (2001) Biochemistry 40, 13407-13412], we showed that 2 equiv of the quinone analogue 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) could occupy the Q(o) site of the cytochrome (cyt) b(6)f complex simultaneously. In this work, a study of electron paramagnetic resonance (EPR) spectra from the oriented cyt b(6)f complex shows that the Rieske iron-sulfur protein (ISP) is in distinct orientations, depending on the stoichiometry of the inhibitor at the Q(o) site. With a single DBMIB at the Q(o) site, the ISP is oriented with the 2Fe-2S cluster toward cyt f, which is similar to the orientation of the ISP in the X-ray crystal structure of the cyt b(6)f complex from thermophilic cyanobacterium Mastigocladus laminosus in the presence of DBMIB, as well as that of the chicken mitochondrial cyt bc(1) complex in the presence of the class II inhibitor myxothiazol, which binds in the so-called "proximal niche", near the cyt b(L) heme. These data suggest that the high-affinity DBMIB site is at the proximal niche Q(o) pocket. With >or=2 equiv of DBMIB bound, the Rieske ISP is in a position that resembles the ISP(B) position of the chicken mitochondrial cyt bc(1) complex in the presence of stigmatellin and the Chlamydomonas reinhardtii cyt b(6)f complex in the presence of tridecylstigmatellin (TDS), which suggests that the low-affinity DBMIB site is at the distal niche. The close interaction of DBMIB bound at the distal niche with the ISP induced the well-known effects on the 2Fe-2S EPR spectrum and redox potential. To further test the effects of DBMIB on the ISP, the extents of cyt f oxidation after flash excitation in the presence of photosystem II inhibitor DCMU were measured as a function of DBMIB concentration in thylakoids. Addition of DBMIB concentrations at which a single binding was expected did not markedly affect the extent of cyt f oxidation, whereas higher concentrations, at which double occupancy was expected, increased the extent of cyt f oxidation to levels similar to that of cyt f oxidation in the presence of a saturating concentration of stigmatellin. Simulations of the EPR g-tensor orientations of the 2Fe-2S cluster versus the physical orientations based on single-crystal studies of the cyt bc(1) complex suggest that the soluble ISP domain of the spinach cyt b(6)f complex can rotate by at least 53 degrees, which is consistent with long-range ISP domain movement. Implications of these results are discussed in the context of the X-ray crystal structures of the chicken mitochondrial cyt bc(1) complex and the M. laminosus and C. reinhardtii cyt b(6)f complexes.     

The effect of pH, ubiquinone depletion and myxothiazol on the reduction kinetics of the prosthetic groups of ubiquinol:cytochrome c oxidoreductase

(1) The kinetics of the reduction by duroquinol of the prosthetic groups of QH2:cytochrome c oxidoreductase and of the formation of ubisemiquinone have been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) The formation of the antimycin-sensitive ubisemiquinone anion parallels the reduction of both high-potential and low-potential cytochrome b-562. (3) The rates of reduction of both the [2Fe-2S] clusters and cytochromes (c + c1) are pH dependent. There is, however, a pH-dependent discrepancy between their rate of reduction, which can be correlated with the difference in pH dependencies of their midpoint potentials. (4) Lowering the pH or the Q content results in a slower reduction of part of the [2Fe-2S] clusters. It is suggested that one cluster is reduced by a quinol/semiquinone couple and the other by a semiquinone/quinone couple. (5) Myxothiazol inhibits the reduction of the [2Fe-2S] clusters, cytochrome c1 and high-potential cytochrome b-562. (6) The results are consistent with a Q-cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase.     

Inhibitor "double occupancy" in the Q(o) pocket of the chloroplast cytochrome b6f complex.

Electron paramagnetic resonance (EPR) spectra of the "Rieske" 2Fe-2S cluster revealed that two molecules of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) can bind to each monomer of the spinach cytochrome (cyt) b6f complex, both in isolated form and in intact thylakoid membranes. Binding to the high-affinity site, which accounts for the observed inhibitory effects, caused small shifts in the g(x) transition of the 2Fe-2S cluster EPR spectrum, similar to those induced by stigmatellin or 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether (DNP-INT). Occupancy of the low-affinity site was only observed after addition of superstoichiometric amounts of the inhibitor and was accompanied by the appearance of a g = 1.94 EPR signal. The shape of the equilibrium binding titration curve, the effects on the 2Fe-2S EPR spectrum, and the ability of the DBMIB binding to displace DNP-INT were consistent with two molecules of DBMIB binding at the Q(o) pocket, with the strongly binding species binding close to the 2Fe-2S cluster. Possible implications of these findings for so-called "double-occupancy" models for Q(o) site catalysis are discussed.     

Proteolytic cleavage of the Fe-S subunit hinge region of Rhodobacter capsulatus bc(1) complex: effects of inhibitors and mutations

The three-dimensional structure of the mitochondrial bc(1) complex reveals that the extrinsic domain of the Fe-S subunit, which carries the redox-active [2Fe2S] cluster, is attached to its transmembrane anchor domain by a short flexible hinge sequence (amino acids D43 to S49 in Rhodobacter capsulatus). In various structures, this extrinsic domain is located in different positions, and the conformation of the hinge region is different. In addition, proteolysis of this region has been observed previously in a bc(1) complex mutant of R. capsulatus [Saribas, A. S., Valkova-Valchanova, M. B., Tokito, M., Zhang, Z., Berry E. A., and Daldal, F. (1998) Biochemistry 37, 8105-8114]. Thus, possible correlations between proteolysis, conformation of the hinge region, and position of the extrinsic domain of the Fe-S subunit within the bc(1) complex were sought. In this work, we show that thermolysin, or an endogenous activity present in R. capsulatus, cleaves the hinge region of the Fe-S subunit between its amino acid residues A46-M47 or D43-V44, respectively, to yield a protease resistant fragment with a M(r) of approximately 18 kDa. The cleavage was affected significantly by ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) site inhibitors and by specific mutations located in the bc(1) complex. In particular, using either purified or detergent dispersed chromatophore-embedded R. capsulatus bc(1) complex, we demonstrated that while stigmatellin blocked the cleavage, myxothiazol hardly affected it, and antimycin A greatly enhanced it. Moreover, mutations in various regions of the Fe-S subunit and cyt b subunit changed drastically proteolysis patterns, indicating that the structure of the hinge region of the Fe-S subunit was modified in these mutants. The overall findings establish that protease accessibility of the Fe-S subunit of the bc(1) complex is a useful biochemical assay for probing the conformation of its hinge region and for monitoring indirectly the position of its extrinsic [2Fe2S] cluster domain within the Q(o) pocket.     

The raised midpoint potential of the [2Fe2S] cluster of cytochrome bc1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit

We have previously reported that mutant strains of Rhodobacter capsulatus that have alanine insertions (+nAla mutants) in the hinge region of the iron sulfur (Fe-S) containing subunit of the bc(1) complex have increased redox midpoint potentials (E(m)) for their [2Fe2S] clusters. The alteration of the E(m) in these strains, which contain mutations far from the metal binding site, implied that the local environment of the metal center is indirectly altered by a change in the interaction of this subunit with the hydroquinone oxidizing (Q(o)) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol. Chem. 277, 3464-3470]. Subsequently, the E(m) changes have been proposed to be predominantly due to a stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Q(o) site inhabitant ubiquinone (Q) [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002) Biochemistry 41, 14372-14382]. To further investigate this issue, Fe-S protein-Q interactions were monitored by electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that the wild type and mutant proteins interactions with Q are similar. Moreover, when the Q(pool) was chemically depleted, the E(m) of the [2Fe2S] cluster in mutant bc(1) complexes remained more positive than a similarly treated native enzyme (e.g., the [2Fe2S] E(m) of the +2Ala mutant was 55 mV more positive than the wild type). These data suggest that the increased E(m) of the [2Fe2S] cluster in the +nAla mutants is in part due to the cluster's interaction with Q, and in part to additional factors that are independent of hydrogen bonding to Q. One such factor, the possibility of a different position of the Fe-S at the Q(o) site of the mutant proteins versus the native enzyme, was addressed by determining the orientation of the [2Fe2S] cluster in the membrane using EPR spectroscopy. In the case of the +2Ala mutant, the [2Fe2S] cluster orientation in the absence of inhibitor is different than that seen in the native enzyme. However, the +2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposed to either stigmatellin or myxothiazol. In addition, Q(pool) extracted membranes of +2Ala mutant exhibited fewer overall orientations, with the predominant one being more similar to that observed in the non-Q-depleted membranes of the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore, additional component(s) that are independent of Q(o) site inhabitants and that originate from the newly observed orientations of the [2Fe2S] clusters in the +nAla mutants also contribute to the increased midpoint potentials of their [2Fe2S] clusters. While the molecular basis of these components remains to be determined, salient implications of these findings in terms of Q(o) site catalysis are discussed.     

The interaction of the Rieske iron-sulfur protein with occupants of the Qo-site of the bc1 complex, probed by electron spin echo envelope modulation.

The bifurcated reaction at the Q(o)-site of the bc(1) complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc(1) complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N(delta) atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N(delta) involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q(o)-site occupant.     

Certain metal ions are inhibitors of cytochrome b6f complex 'Rieske' iron-sulfur protein domain movements

Many current models of the Q cycle for the cytochrome (cyt) b6f and the cyt bc1 complexes incorporate 'Rieske' iron-sulfur protein (ISP) domain movements to gate electron transfer and to ensure high yields of proton shuttling. It was previously proposed that copper ions, which bind at a site distant from the quinol oxidase (Q(o)) site, inhibit plastoquinol (PQH2) binding by restraining the hydrophilic head domain of the ISP [Rao B. K., S., Tyryshkin, A. M., Roberts, A. G., Bowman, M. K., and Kramer, D. M. (1999) Biochemistry 38, 3285-3296]. The present work presents evidence that this is indeed the case for both copper ions and Zn2+, which appear to inhibit by similar mechanisms. Electron paramagnetic resonance (EPR) spectra show that Cu2+ and Zn2+ binding to the cyt b6f complex displaces the Q(o) site inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). At high concentrations, both DBMIB and Cu2+ or Zn2+ can bind simultaneously, altering the Rieske 2Fe2S cluster and Cu2+ EPR spectra, suggesting perturbations in their respective binding sites. Both Zn2+ and Cu1+ altered the orientations of the Rieske 2Fe2S cluster with respect to the membrane plane, but had no effect on that of the cyt b6 hemes. Cu2+ was found to change the orientation of the cyt f heme plane, consistent with binding on the cyt f protein. Within conservative constraints, the data suggest that the ISP is shifted into a position intermediate between the ISP(C) position, when the Q(o) site is unoccupied, and the ISP(B) position, when the Q(o) site is occupied by inhibitors such as DBMIB or stigmatellin. These results support the role of ISP domain movements in Q(o) site catalysis.     

The pathway of electrons through OH2:cytochrome c oxidoreductase studied by pre-steady -state kinetics

The kinetic behaviour of the prosthetic groups and the semiquinones in in QH2:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Qin, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. the two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2S] clusters and cytochrome c1 are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Qin is formed and both the Fe-S clusters and cytochrome c1 are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Qout (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996-11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase is proposed.     

Characterization of the iron-sulfur cluster N7 (N1c) in the subunit NuoG of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX(27)C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated Delta N1b, Delta N1c, Delta N4, and Delta N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g(x,y,z) = 1.89, 1.94, and 2.06, and g(x,y,z) = 1.91, 1.94, and 2.05 at 6-20 K in wild type, Delta N1b, and Delta N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in Delta N4 and Delta N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knock-out strain, the implicated structural importance of the iron-sulfur binding domains is discussed.     

Ubiquinone binding capacity of the Rhodobacter capsulatus cytochrome bc1 complex: effect of diphenylamine, a weak binding QO site inhibitor

Diphenylamine (DPA), a known inhibitor of polyene and isoprene biosynthesis, is shown to inhibit flash-activatable electron transfer in photosynthetic membranes of Rhodobacter capsulatus. DPA is specific to the QO site of ubihydroquinone:cytochrome c oxidoreductase, where it inhibits not only reduction of the [2Fe-2S]2+ cluster in the FeS subunit and subsequent cytochrome c reduction but also heme bL reduction in the cytochrome b subunit. In both cases, the kinetic inhibition constant (Ki) is 25 +/- 10 microM. A novel aspect of the mode of action of DPA is that complete inhibition is established without disturbing the interaction between the reduced [2Fe-2S]+ cluster and the QO site ubiquinone complement, as observed from the electron paramagnetic resonance (EPR) spectral line shape of the reduced [2Fe-2S] cluster, which remained characteristic of two ubiquinones being present. These observations imply that DPA is behaving as a noncompetitive inhibitor of the QO site. Nevertheless, at higher concentrations (>10 mM), DPA can interfere with the QO site ubiquinone occupancy, leading to a [2Fe-2S] cluster EPR spectrum characteristic of the presence of only one ubiquinone in the QO site. Evidently, DPA can displace the more weakly bound of the two ubiquinones in the site, but this is not requisite for its inhibiting action.     

The Q cycle of cytochrome bc complexes: a structure perspective

Aspects of the crystal structures of the hetero-oligomeric cytochrome bc(1) and b(6)f ("bc") complexes relevant to their electron/proton transfer function and the associated redox reactions of the lipophilic quinones are discussed. Differences between the b(6)f and bc(1) complexes are emphasized. The cytochrome bc(1) and b(6)f dimeric complexes diverge in structure from a core of subunits that coordinate redox groups consisting of two bis-histidine coordinated hemes, a heme b(n) and b(p) on the electrochemically negative (n) and positive (p) sides of the complex, the high potential [2Fe-2S] cluster and c-type heme at the p-side aqueous interface and aqueous phase, respectively, and quinone/quinol binding sites on the n- and p-sides of the complex. The bc(1) and b(6)f complexes diverge in subunit composition and structure away from this core. b(6)f Also contains additional prosthetic groups including a c-type heme c(n) on the n-side, and a chlorophyll a and β-carotene. Common structure aspects; functions of the symmetric dimer. (I) Quinone exchange with the bilayer. An inter-monomer protein-free cavity of approximately 30? along the membrane normal×25? (central inter-monomer distance)×15? (depth in the center), is common to both bc(1) and b(6)f complexes, providing a niche in which the lipophilic quinone/quinol (Q/QH(2)) can be exchanged with the membrane bilayer. (II) Electron transfer. The dimeric structure and the proximity of the two hemes b(p) on the electrochemically positive side of the complex in the two monomer units allow the possibility of two alternate routes of electron transfer across the complex from heme b(p) to b(n): intra-monomer and inter-monomer involving electron cross-over between the two hemes b(p). A structure-based summary of inter-heme distances in seven bc complexes, representing mitochondrial, chromatophore, cyanobacterial, and algal sources, indicates that, based on the distance parameter, the intra-monomer pathway would be favored kinetically. (III) Separation of quinone binding sites. A consequence of the dimer structure and the position of the Q/QH(2) binding sites is that the p-side QH(2) oxidation and n-side Q reduction sites are each well separated. Therefore, in the event of an overlap in residence time by QH(2) or Q molecules at the two oxidation or reduction sites, their spatial separation would result in minimal steric interference between extended Q or QH(2) isoprenoid chains. (IV) Trans-membrane QH(2)/Q transfer. (i) n/p-side QH(2)/Q transfer may be hindered by lipid acyl chains; (ii) the shorter less hindered inter-monomer pathway across the complex would not pass through the center of the cavity, as inferred from the n-side antimycin site on one monomer and the p-side stigmatellin site on the other residing on the same surface of the complex. (V) Narrow p-side portal for QH(2)/Q passage. The [2Fe-2S] cluster that serves as oxidant, and whose histidine ligand serves as a H(+) acceptor in the oxidation of QH(2), is connected to the inter-monomer cavity by a narrow extended portal, which is also occupied in the b(6)f complex by the 20 carbon phytyl chain of the bound chlorophyll.     

Architecture of the Qo site of the cytochrome bc1 complex probed by superoxide production

Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.     

The membrane-bound [NiFe]-hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron-sulphur clusters.

The purified membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron-sulphur clusters. The EPR spectra showed signals from three different [4Fe-4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni-Fe site indicated that this was the proximal cluster as found in all [NiFe]-hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four-Cys motifs for the binding of a [4Fe-4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH-dependent midpoint potential. The possible relevance of these properties for the function of this proton-pumping [NiFe]-hydrogenase is discussed.     

Proton-coupled electron transfer at the Qo-site of the bc1 complex controls the rate of ubihydroquinone oxidation

The rate-limiting reaction of the bc(1) complex from Rhodobacter sphaeroides is transfer of the first electron from ubihydroquinone (quinol, QH(2)) to the [2Fe-2S] cluster of the Rieske iron-sulfur protein (ISP) at the Q(o)-site. Formation of the ES-complex requires participation of two substrates (S), QH(2) and ISP(ox). From the variation of rate with [S], the binding constants for both substrates involved in formation of the complex can be estimated. The configuration of the ES-complex likely involves the dissociated form of the oxidized ISP (ISP(ox)) docked at the b-interface on cyt b, in a complex in which N(epsilon) of His-161 (bovine sequence) forms a H-bond with the quinol -OH. A coupled proton and electron transfer occurs along this H-bond. This brief review discusses the information available on the nature of this reaction from kinetic, structural and mutagenesis studies. The rate is much slower than expected from the distance involved, likely because it is controlled by the low probability of finding the proton in the configuration required for electron transfer. A simplified treatment of the activation barrier is developed in terms of a probability function determined by the Br?nsted relationship, and a Marcus treatment of the electron transfer step. Incorporation of this relationship into a computer model allows exploration of the energy landscape. A set of parameters including reasonable values for activation energy, reorganization energy, distances between reactants, and driving forces, all consistent with experimental data, explains why the rate is slow, and accounts for the altered kinetics in mutant strains in which the driving force and energy profile are modified by changes in E(m) and/or pK of ISP or heme b(L).     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.