Superoxide anion generation by the cytochrome bc1 complex

We have measured the rates of superoxide anion generation by cytochrome bc₁ complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc₁ complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc₁ complexes the rate of superoxide anion production was greatest in the absence of bc₁ inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc₁ complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc₁ complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc₁ complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.     

The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex

Summary A cytochrome bc ₁-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc ₁-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c ₁. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c ₁ is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.Abbreviations BSA bovine serum albumin - SDS sodium dodecylsulphate - Rs Rhodospirillum - Rb Rhodobacter - Pc Paracoccus - Rps Rhodopseudomonas The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X55387     

Role of the Rieske Iron–Sulfur Protein Midpoint Potential in the Protonmotive Q-Cycle Mechanism of the Cytochrome bc 1 Complex

The midpoint potential of the [2Fe–2S] cluster of the Rieske iron–sulfurprotein (E _{m 7} = +280mV) is the primary determinant of the rate of electron transfer from ubiquinol to cytochromec catalyzed by the cytochrome bc ₁ complex. As the midpoint potential of the Rieske clusteris lowered by altering the electronic environment surrounding the cluster, theubiquinol-cytochrome c reductase activity of the bc ₁ complex decreases; between 220 and 280 mV therate changes 2.5-fold. The midpoint potential of the Rieske cluster also affects thepresteady-state kinetics of cytochrome b and c ₁ reduction. When the midpoint potential of the Rieskecluster is more positive than that of the heme of cytochrome c ₁, reduction of cytochrome bis biphasic. The fast phase of b reduction is linked to the optically invisible reduction of theRieske center, while the rate of the second, slow phase matches that of c ₁ reduction. The ratesof b and c ₁ reduction become slower as the potential of the Rieske cluster decreases andchange from biphasic to monophasic as the Rieske potential approaches that of theubiquinone/ubiquinol couple. Reduction of b and c ₁ remain kinetically linked as the midpoint potentialof the Rieske cluster is varied by 180 mV and under conditions where the presteady statereduction is biphasic or monophasic. The persistent linkage of the rates of b and c ₁ reduction isaccounted for by the bifurcated oxidation of ubiquinol that is unique to the Q-cycle mechanism.     

Characterization of the pet operon of Rhodospirillum rubrum

The three genes of the pet operon, coding, respectively, for the Rieske iron-sulfur protein, cytochrome b and cytochrome c ₁ components of the cytochrome bc ₁ complex in the photosynthetic bacterium Rhodospirillum rubrum have been sequenced. The amino acid sequences deduced for these three peptides from the nucleotide sequences of the genes have been confirmed, in part, by direct sequencing of portions of the three peptides separated from a sample of the purified, detergent-solubilized complex. These sequences show considerable homology with those previously obtained for the pet operons of other photosynthetic bacteria. Northern blots of R. rubrum mRNA have established that the operon is transcribed as a single polycistronic message, the start site of which has been determined by both primer extension and nuclease protection. Photosynthetic growth of R. rubrum was shown to be inhibited by antimycin A, a specific inhibitor of cytochrome bc ₁ complexes, and antimycin A-resistant mutants of R. rubrum have been isolated. Preliminary results suggest that it may be possible to express the R. rubrum pet operon in a strain of the photosynthetic bacterium Rhodobacter capsulatus from which the native pet operon has been deleted.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

Integration of the Mitochondrial-Processing Peptidase into the Cytochrome bc 1 Complex in Plants

The plant mitochondrial cytochrome bc ₁ complex, like nonplant mitochondrial complexes,consists of cytochromes b and c ₁, the Rieske iron–sulfur protein, two Core proteins, and fivelow-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteinsare identical to subunits of the general mitochondrial processing peptidase (MPP). The MPPis a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptidesfrom hundreds of the nuclear-encoded mitochondrial precursor proteins that are synthesizedin the cytosol and imported into the mitochondrion. Integration of the MPP into the bc ₁complex renders the bc ₁ complex in plants bifunctional, being involved both in electrontransport and in protein processing. Despite the integration of MPP into the bc ₁ complex,electron transfer as well as translocation of the precursor through the import channel areindependent of the protein-processing activity. Recognition of the processing site by MPPoccurs via the recognition of higher-order structural elements in combination with charge andcleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammaliancytochrome bc ₁ complex is highly useful for understanding of the mechanism of action of MPP.In memory of my teacher—an insightful, devoted, and enthusiastic scientist and an amiable and kind-hearted human being—Lars Ernster     

Assembly of Deletion Mutants of the Rieske Iron–Sulfur Protein into the Cytochrome bc 1 Complex of Yeast Mitochondria

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc ₁ complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc ₁ complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc ₁ complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc ₁ complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc ₁ complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc ₁ complex; however, this association did not lead to enzymatic activity of the bc ₁ complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.     

The three-subunit cytochromebc 1 complex ofParacoccus denitrificans. Its physiological function,structure, and mechanism of electron transfer and energy transduction

The cytochromebc ₁ complex purified fromP. denitrificans has the same electron-transfer and energy-transducing activities, is sensitive to the same electron-transfer inhibitors, and contains cytochromesb, c ₁, iron-sulfur protein, and thermodynamically stable ubisemiquinone identical to the counterpart complexes from mitochondria. However, the bacterialbc ₁ complex consists of only three proteins, the obligate electron-transfer proteins, while the mitochondrial complexes contain six or more supernumerary poly-peptides, which have no obvious electron-transfer function. TheP. denitrificans complex is a paradigm for thebc ₁ complexes of all gram-negative bacteria. In addition, because of its simple polypeptide composition and apparently minimal damage during isolation, theP. denitrificans bc ₁ complex is an ideal system in which to study structure-function relationships requisite to energy transduction linked to electron transfer.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

Structure and Function of the Bacterial bc 1 Complex: Domain Movement, Subunit Interactions, and Emerging Rationale Engineering Attempts

The ubiquinol: cytochrome c oxidoreductase, or the bc ₁ complex, is a key component ofboth respiratory and photosynthetic electron transfer and contributes to the formation of anelectrochemical gradient necessary for ATP synthesis. Numerous bacteria harbor a bc ₁ complexcomprised of three redox-active subunits, which bear two b-type hemes, one c-type heme, andone [2Fe–2S] cluster as prosthetic groups. Photosynthetic bacteria like Rhodobacter speciesprovide powerful models for studying the function and structure of this enzyme and are beingwidely used. In recent years, extensive use of spontaneous and site-directed mutants and theirrevertants, new inhibitors, discovery of natural variants of this enzyme in various species, andengineering of novel bc ₁ complexes in species amenable to genetic manipulations have providedus 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 variousmitochondrial bc ₁ complexes in different crystallographic forms has consolidated previousfindings, added atomic-scale precision to our knowledge, and raised new issues, such as thepossible movement of the Rieske Fe–S protein subunit during Q_o site catalysis. Here, studiesperformed during the last few years using bacterial bc ₁ complexes are reviewed briefly andongoing investigations and future challenges of this exciting field are mentioned.     

Thebc 1 complexes ofRhodobacter sphaeroides andRhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochromec oxidoreductase (bc ₁ complex). In bothRhodobacter sphaeroides andRhodobacter capsulatus, thebc ₁ complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because thebc ₁ complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochromeb subunit, in the Rieske iron-sulfur subunit, and in the cytochromec ₁ subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q_o) site or the quinol reductase (Q_i) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.     

Role of the Twin Arginine Protein Transport Pathway in the Assembly of the Streptomyces coelicolor Cytochrome bc1 Complex

The cytochrome bc₁-cytochrome aa₃ complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc₁ complex shows a number of unusual features in comparison to other cytochrome bc₁ complexes. In particular, the Rieske iron-sulfur protein component of this complex, QcrA, is a polytopic rather than a monotopic membrane protein. Bacterial Rieske proteins are usually integrated into the membrane in a folded conformation by the twin arginine protein transport (Tat) pathway. In this study, we show that the activity of the Streptomyces coelicolor M145 cytochrome bc₁ complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a ΔtatC mutant strain, indicating that a second protein translocation machinery also participates in its assembly. Difference spectroscopy indicated that the cytochrome c component of the complex was correctly assembled in the absence of the Tat machinery. We show that the intact cytochrome bc₁ complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc₁ complex containing the Rieske protein can be isolated from membranes even when the Tat system is inactive. These findings strongly suggest that the additional transmembrane segments of the S. coelicolor Rieske protein mediate hydrophobic interactions with one or both of the cytochrome subunits.     

Cytochrome bc 1 and b 6 f complexes of photosynthetic membranes

All photosynthetic membranes contain a cytochrome bc ₁ or b ₆ f complex that catalyzes the oxidation of quinols and the reduction of a high-potential electron carrier, such as cytochrome c ₂ or plastocyanin. The cytochrome complex also functions in the translocation of protons across the membrane and as a consequence, establishes the proton motive force that is used for the synthesis of ATP. The structure and function of the cytochrome complexes are first reviewed in this chapter. Amino acid sequence information for almost all of the protein subunits of these complexes is now available, and these allow for a detailed consideration of functional domains in the protein subunits and for a further discussion of the evolution of the cytochrome complex in photosynthetic organisms.     

Strong homology between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase of two species of Acetabularia and the occurrence of unusual codon usage

Summary The complete nucleotide sequence of the genes encoding the Rieske FeS, the cytochrome b and the cytochrome c ₁ subunits of the ubiquinol-cytochrome c ₂ oxidoreductase from the photosynthetic purple bacterium Rhodopseudomonas viridis, and the derived amino acid sequences are presented. These three genes, fbcF, fbcB and fbcC, are located at contiguous sites of the genome. The DNA-deduced amino acid sequences are compared with known primary structures of corresponding proteins from other purple photosynthetic bacteria, as well as mitochondria, cyanobacteria and chloroplasts.Abbreviations BSA bovine serum albumin - Rb Rhodobacter - Rps Rhodopseudomonas     

Molecular modeling studies of the DCCD-treated cytochrome bc1 complex: predicted conformational changes and inhibition of proton translocation

Dicyclohexylcarbodiimide (DCCD) binds covalently to an acidic amino acid located in the cd loop connecting membrane-spanning helices C and D of cytochrome b resulting in an inhibition of proton translocation in the cytochrome bc ₁ complex with minimal effects on the steady state rate of electron transfer. Single turnover studies performed with the yeast cytochrome bc ₁ complex indicated that the initial phase of cytochrome b reduction was inhibited 25–45% in the DCCD-treated cytochrome bc ₁ complex, while the rate of cytochrome c ₁ reduction was unaffected. Simulations by molecular modeling predict that binding of DCCD to glutamate 163 located in the cd2 loop of cytochrome b of chicken liver mitochondria results in major conformational changes in the protein. The conformation of the cd loop and the end of helix C appeared twisted with a concomitant rearrangement of the amino acid residues of both cd1 and cd2 loops. The predicted rearrangement of the amino acid residues of the cd loop results in disruptions of the hydrogen bonds predicted to form between amino acid residues of the cd and ef loops. Simultaneously, two new hydrogen bonds are predicted to form between glutamate 272 and two residues, aspartate 253 and tyrosine 272. Formation of these new hydrogen bonds would restrict the rotation and protonation of glutamate 272, which may be necessary for the release of the second electrogenic proton obtained during ubiquinol oxidation in the bc₁ complex.     

Structural Basis of Multifunctional Bovine Mitochondrial Cytochrome bc 1 Complex

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

Functional characterization of the lesion in the ubiquinol: Cytochromec oxidoreductase complex isolated from the nonphotosynthetic strain R126 ofRhodobacter capsulatus

The cytochromebc ₁ complexes from the nonphotosynthetic strain R126 ofRhodobacter capsulatus and from its revertant MR126 were purified. Between both preparations, no difference could be observed in the stoichiometries of the cytochromes, in their spectral properties, and in their midpoint redox potentials. Both also showed identical polypeptide patterns after electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulfate. The ubiquinol: cytochromec oxidoreductase activity was strongly inhibited in the complex from the mutant compared to the one from the revertant. So was the oxidant-induced extra reduction of cytochromeb. Both preparations, however, showed an antimycin-induced red shift of cytochromeb, as well as antimycin-sensitive reduction of cytochromeb by ubiquinol. In accordance with a preceding study of chromatophores (Robertsonet al. (1986).J. Biol. Chem. 261, 584–591), it is concluded that the mutation affects specifically the ubiquinol oxidizing site, leaving the ubiquinol reducing site unchanged.     

The general mitochondrial processing peptidase from wheat is integrated into the cytochrome bc 1-complex of the respiratory chain

The bc ₁-complex (EC 1.10.2.2.) from Triticum aestivum L. was purified by cytochrome-c affinity chromatography and gel filtration using either etiolated seedlings or wheat-germ extract as starting material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the isolated enzyme revealed ten bands, which were analysed by immunoblotting and direct amino-acid sequencing. The enzyme from wheat is the first bc ₁-complex that is reported to contain four core proteins (55.5, 55.0, 51.5 and 51.0 kDa). In addition, the wheat bc ₁-complex comprises cytochrome b (35 kDa), cytochrome c ₁ (33 kDa) the Rieske iron-sulphur protein (25 kDa) and three small subunits < 15 kDa. This composition differs from the one reported in fungi, mammals and potato. Partial sequence determination of the large subunits suggests that the 55.5 and 55.0-kDa-proteins represent the -subunit of the general mitochondrial processing peptidase, and the 51.5 and 51.0-kDa proteins the -subunit of this enzyme. The bc ₁-complex from wheat efficiently processes mitochondrial precursor proteins as shown in an in-vitro processing assay. In control experiments the isolated bc ₁-complexes from potato, yeast, Neurospora and beef, all purified by the same isolation procedure, were also tested for processing activity. Only the protein complexes from plants contain the general mitochondrial processing peptidase. The composition of the wheat bc ₁-complex sheds new light on the co-evolution of the processing peptidase and the middle segment of the respiratory chain.Abbreviations MPP mitochondrial processing peptidase We wish to thank Prof. G. Schatz, Biozentrum Basel, Switzerland and Prof. H. Weiss, Universität Düsseldorf, Germany for providing antibodies against the repiratory subunits of the bc ₁-complex from yeast and Neurospora and to H. Mentzel, A. Leisse, R. Breitfeld and B. Hidde for excellent technical assistance. Thanks are also due to Prof. M. Boutry, Université de Louvaine-la-Neuve, Belgium for providing a plasmid containing the -subunit of ATPase from tobacco. This research was supported by the Deutsche Forschungsgemeinschalft and the Bundesministerium für Forschung und Technologie.