The 2.6 A resolution structure of Rhodobacter capsulatus bacterioferritin with metal-free dinuclear site and heme iron in a crystallographic 'special position'.

Bacterioferritin from Rhodobacter capsulatus was crystallized and its structure was solved at 2.6 A resolution. This first structure of a bacterioferritin from a photosynthetic organism is a spherical particle of 24 subunits displaying 432 point-group symmetry like ferritin and bacterioferritin from Escherichia coli. Crystallized in the I422 space group, its structural analysis reveals for the first time the non-symmetric heme molecule located on a twofold crystallographic symmetry axis. Other hemes of the protomer are situated on twofold noncrystallographic axes. Apparently, both types of sites bind heme in two orientations, leading to an average structure consisting of a symmetric 50:50 mixture, thus satisfying the crystallographic and noncrystallographic symmetry of the crystal. Five water molecules are situated close to the heme, which is bound in a hydrophobic pocket and axially coordinated by two crystallographic or noncrystallographically related methionine residues. Its ferroxidase center, in which Fe(II) is oxidized to Fe(III), is empty or fractionally occupied by a metal ion. Two positions are observed for the coordinating Glu18 side chain instead of one in the E. coli enzyme in which the site is occupied. This result suggests that the orientation of the Glu18 side chain could be constrained by this interaction.

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.

The RegB/RegA two-component regulatory system controls synthesis of photosynthesis and respiratory electron transfer components in Rhodobacter capsulatus.

Recently, we demonstrated that the RegB/RegA two-component regulatory system from Rhodobacter capsulatus functions as a global regulator of metabolic processes that either generate or consume reducing equivalents. For example, the RegB/RegA system controls expression of such energy generating processes as photosynthesis and hydrogen utilization. In addition, RegB/RegA also control nitrogen and carbon fixation pathways that utilize reducing equivalents. Here, we use a combination of DNase I protection and plasmid-based reporter expression studies to demonstrate that RegA directly controls synthesis of cytochrome cbb3 and ubiquinol oxidases that function as terminal electron acceptors in a branched respiratory chain. We also demonstrate that RegA controls expression of cytochromes c2, c(y) and the cytochrome bc1 complex that are involved in both photosynthetic and respiratory electron transfer events. These data provide evidence that the RegB/RegA two-component system has a major role in controlling the synthesis of numerous processes that affect reducing equivalents in Rhodobacter capsulatus.

Large scale domain movement in cytochrome bc(1): a new device for electron transfer in proteins.

Recently, crystallographic, spectroscopic, kinetic and biochemical genetic data have merged to unveil a large domain movement for the Fe-S subunit in cytochrome bc(1). In this evolutionarily conserved enzyme, the domain motion acts to conduct intra-complex electron transfer and is essential for redox energy conversion.

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides.

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.

Probing the role of the Fe-S subunit hinge region during Q(o) site catalysis in Rhodobacter capsulatus bc(1) complex.

The ubihydroquinone:cytochrome c oxidoreductase, or bc(1) complex, functions according to a mechanism known as the modified Q cycle. Recent crystallographic data have revealed that the extrinsic domain containing the [2Fe2S] cluster of the Fe-S subunit of this enzyme occupies different positions in various crystal forms, suggesting that this subunit may move during ubihydroquinone oxidation. As in these structures the hydrophobic membrane anchor of the Fe-S subunit remains at the same position, the movement of the [2Fe2S] cluster domain would require conformational changes of the hinge region linking its membrane anchor to its extrinsic domain. To probe the role of the hinge region, Rhodobacter capsulatus bc(1) complex was used as a model, and various mutations altering the hinge region amino acid sequence, length, and flexibility were obtained. The effects of these modifications on the bc(1) complex function and assembly were investigated in detail. These studies demonstrated that the nature of the amino acid residues located in the hinge region (positions 43-49) of R. capsulatus Fe-S subunit was not essential per se for the function of the bc(1) complex. Mutants with a shorter hinge (up to five amino acid residues deletion) yielded functional bc(1) complexes, but contained substoichiometric amounts of the Fe-S subunit. Moreover, mutants with increased rigidity or flexibility of the hinge region altered both the function and the assembly or the steady-state stability of the bc(1) complex. In particular, the extrinsic domain of the Fe-S subunit of a mutant containing six proline residues in the hinge region was shown to be locked in a position similar to that seen in the presence of stigmatellin. Interestingly, the latter mutant readily overcomes this functional defect by accumulating an additional mutation which shortens the length of the hinge. These findings indicate that the hinge region of the Fe-S subunit of bacterial bc(1) complexes has a remarkable structural plasticity.

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.

Uncovering the [2Fe2S] domain movement in cytochrome bc1 and its implications for energy conversion.

In crystals of the key respiratory and photosynthetic electron transfer protein called ubihydroquinone:cytochrome (cyt) c oxidoreductase or cyt bc(1), the extrinsic [2Fe2S] cluster domain of its Fe-S subunit assumes several conformations, suggesting that it may move during catalysis. Herein, using Rhodobacter capsulatus mutants that have modifications in the hinge region of this subunit, we were able to reveal this motion kinetically. Thus, the bc(1) complex (and possibly the homologous b(6)f complex in chloroplasts) employs the [2Fe2S] cluster domain as a device to shuttle electrons from ubihydroquinone to cyt c(1) (or cyt f). We demonstrate that this domain movement is essential for cyt bc(1) function, because a mutant enzyme with a nonmoving Fe-S subunit has no catalytic activity, and one with a slower movement has lower activity. This motion is apparently designed with a natural frequency slow enough to assure productive Q(o) site charge separation but fast enough not to be rate limiting. These findings add the unprecedented function of intracomplex electron shuttling to large-scale domain motions in proteins and may well provide a target for cyt bc(1) antibiotics.

Roles of the ccoGHIS gene products in the biogenesis of the cbb(3)-type cytochrome c oxidase.

In many bacteria the ccoGHIS cluster, located immediately downstream of the structural genes (ccoNOQP) of cytochrome cbb(3) oxidase, is required for the biogenesis of this enzyme. Genetic analysis of ccoGHIS in Rhodobacter capsulatus demonstrated that ccoG, ccoH, ccoI and ccoS are expressed independently of each other, and do not form a simple operon. Absence of CcoG, which has putative (4Fe-4S) cluster binding motifs, does not significantly affect cytochrome cbb(3) oxidase activity. However, CcoH and CcoI are required for normal steady-state amounts of the enzyme. CcoI is highly homologous to ATP-dependent metal ion transporters, and appears to be involved in the acquisition of copper for cytochrome cbb(3) oxidase, since a CcoI-minus phenotype could be mimicked by copper ion starvation of a wild-type strain. Remarkably, the small protein CcoS, with a putative single transmembrane span, is essential for the incorporation of the redox-active prosthetic groups (heme b, heme b(3 )and Cu) into the cytochrome cbb(3) oxidase. Thus, the ccoGHIS products are involved in several steps during the maturation of the cytochrome cbb(3) oxidase.

Novel Rhodobacter capsulatus genes required for the biogenesis of various c-type cytochromes.

Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c-type cytochrome (cyt)-deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c-type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild-type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram-negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the gram-positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA:phoA and ccdA :lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some gram-negative bacteria such as R. capsulatus.

Structure and function of the bacterial bc1 complex: domain movement, subunit interactions, and emerging rationale engineering attempts.

The ubiquinol: cytochrome c oxidoreductase, or the bc1 complex, is a key component of both respiratory and photosynthetic electron transfer and contributes to the formation of an electrochemical gradient necessary for ATP synthesis. Numerous bacteria harbor a bc1 complex comprised of three redox-active subunits, which bear two b-type hemes, one c-type heme, and one [2Fe-2S] cluster as prosthetic groups. Photosynthetic bacteria like Rhodobacter species provide powerful models for studying the function and structure of this enzyme and are being widely used. In recent years, extensive use of spontaneous and site-directed mutants and their revertants, new inhibitors, discovery of natural variants of this enzyme in various species, and engineering of novel bc1 complexes in species amenable to genetic manipulations have provided us with a wealth of information on the mechanism of function, nature of subunit interactions, and assembly of this important enzyme. The recent resolution of the structure of various mitochondrial bc1 complexes in different crystallographic forms has consolidated previous findings, added atomic-scale precision to our knowledge, and raised new issues, such as the possible movement of the Rieske Fe-S protein subunit during Qo site catalysis. Here, studies performed during the last few years using bacterial bc1 complexes are reviewed briefly and ongoing investigations and future challenges of this exciting field are mentioned.

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.

Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites.

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.

An engineered cytochrome b6c1 complex with a split cytochrome b is able to support photosynthetic growth of Rhodobacter capsulatus.

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.

Substitution of the sixth axial ligand of Rhodobacter capsulatus cytochrome c1 heme yields novel cytochrome c1 variants with unusual properties.

The cytochrome (cyt) c1 heme of the ubihydroquinone:cytochrome c oxidoreductase (bc1 complex) is covalently attached to two cysteine residues of the cyt c1 polypeptide chain via two thioether bonds, and the fifth and sixth axial ligands of its iron atom are histidine (H) and methionine (M), respectively. The latter residue is M183 in Rhodobacter capsulatus cyt c1, and previous mutagenesis studies revealed its critical role for the physicochemical properties of cyt c1 [Gray, K. A., Davidson, E., and Daldal, F. (1992) Biochemistry 31, 11864-11873]. In the homologous chloroplast b6f complex, the sixth axial ligand is provided by the amino group of the amino terminal tyrosine residue. To further pursue our investigation on the role played by the sixth axial ligand in heme-protein interactions, novel cyt c1 variants with histidine-lysine (K) and histidine-histidine axial coordination were sought. Using a R. capsulatus genetic system, the cyt c1 mutants M183K and M183H were constructed by site-directed mutagenesis, and chromatophore membranes as well as purified bc1 complexes obtained from these mutants were characterized in detail. The studies revealed that these mutants incorporated the heme group into the mature cyt c1 polypeptides, but yielded nonfunctional bc1 complexes with unusual spectroscopic and thermodynamic properties, including shifted optical absorption maxima (lambdamax) and decreased redox midpoint potential values (Em7). The availability and future detailed studies of these stable cyt c1 mutants should contribute to our understanding of how different factors influence the physicochemical and folding properties of membrane-bound c-type cytochromes in general.

The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer.

Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs. Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined. It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R. capsulatus counterpart, Cyt cyRc. Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?

Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1.

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.

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.

Isolation and characterization of a two-subunit cytochrome b-c1 subcomplex from Rhodobacter capsulatus and reconstitution of its ubihydroquinone oxidation (Qo) site with purified Fe-S protein subunit.

The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.