Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

The cytochrome bc 1 complexes of photosynthetic purple bacteria

Complete nucleotide sequences are now available for the pet (fbc) operons coding for the three electron carrying protein subunits of the cytochrome bc ₁ complexes of four photosynthetic purple non-sulfur bacteria. It has been demonstrated that, although the complex from one of these bacteria may contain a fourth subunit, three subunit complexes appear to be fully functional. The ligands to the three hemes and the one [2Fe-2S] cluster in the complex have been identified and considerable progress has been made in mapping the two quinone-binding sites present in the complex, as well as the binding sites for quinone analog inhibitors. Hydropathy analyses and alkaline phosphatase fusion experiments have provided considerable insight into the likely folding pattern of the cytochrome b peptide of the complex and identification of the electrogenic steps associated with electron transport through the complex has allowed the orientation within the membrane of the electron-carrying groups of the complex to be modeled.     

Isolation of cytochrome bc 1 complexes from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodospirillum rubrum

Cytochrome bc ₁ complexes have been isolated from wild type Rhodopseudomonas viridis and Rhodospirillum rubrum and purified by affinity chromatography on cytochrome c-Sepharose 4B. Both complexes are largely free of bacteriochlorophyll and carotenoids and contain cytochromes b and c ₁ in a 2:1 molar ratio. For the Rps. viridis complex, evidence has been obtained for two spectrally distinct b-cytochromes. The R. rubrum complex contains a Rieske iron-sulfur protein (present in approximately 1:1 molar ratio to cytochrome c ₁) and catalyzes an antimycin A- and myxothiazol-sensitive electron transfer from duroquinol to equine cytochrome c or R. rubrum cytochrome c ₂. Although an attempt to prepare a cytochrome bc ₁ complex from the gliding green bacterium Chloroflexus aurantiacus was not successful, membranes isolated from phototrophically grown Cfl. aurantiacus were shown to contain a Rieske iron-sulfur protein and protoheme (the prosthetic group of b-type cytochromes).Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb 5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb ₅ performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb ₅ is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.     

The nucleotide sequence of the puf operon from the purple photosynthetic bacterium, Rhodospirillum molischianum: Comparative analyses of light-harvesting proteins and the cytochrome subunits associated with the reaction centers

The nucleotide sequence of the puf operon of the purple bacterium, Rhodospirillum molischianum, was determined. The operon includes genes coding for the and subunits of the light-harvesting 1 (LH1) complex and the L, M, and cytochrome subunits of the reaction center complex. As in other purple bacteria, the genes are arranged within the operon in this order. As in Rubrivivax gelatinosus, the deduced amino acid sequence of the cytochrome subunit in Rsp. molischianum contains significant deletions at the attachment site to the M subunit compared with that of Rhodopseudomonas viridis. This suggests that the interaction between the cytochrome subunit and the LM core in Rsp. molischianum and Rvi. gelatinosus is different from that in Rps. viridis. Phylogenetic analysis of the light-harvesting proteins indicated that the LH1 and subunits of Rsp. molischianum are included in the lineage of LH1 polypeptides of the purple bacteria, while the LH2 and subunits are positioned apart from LH2 polypeptides of the other purple bacteria together with those of Chromatium vinosum. Based on these phylogenetic analyses, the classification of the light-harvesting proteins in purple bacteria is discussed.     

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

Defects in the cytochromebc 1 complex in mitochondrial diseases

The clinical and biochemical findings of 14 patients with an isolated defect of thebc ₁ complex have been summarized. The heterogeneity of this group of disorders reflects the severity and tissue specific expression of the defect and the complexity of this multisubunit protein with components that are coded on both nuclear and mitochondrial DNA. The data on several patients with a combined defect of cytochrome oxidase and thebc ₁ complex or with multiple respiratory chain defects have also been presented and discussed in relation to our knowledge of the biosynthesis and assembly of the respiratory chain complexes. The severity of the defectin vivo is illustrated in one patient with isolated complex III deficiency by measurement of O₂ consumption and CO₂ production following exercise, or by³¹P-NMR. The latter also provides a means by which response to therapy can be followed.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrialbc 1 complex

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrialbc ₁ complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in thebc ₁ complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochromec reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of thebc ₁ complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochromec reductase activity. However, a clear stimulation of the rate of cytochromeb reduction in parallel to an inhibition of cytochromeb oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on thebc ₁ complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.     

Kinetics of Electron Transfer within Cytochrome bc 1 and Between Cytochrome bc 1 and Cytochrome c

In this minireview an overview is presented of the kinetics of electron transfer within the cytochrome bc (1) complex, as well as from cytochrome bc (1) to cytochrome c. The cytochrome bc (1) complex (ubiquinone:cytochrome c oxidoreductase) is an integral membrane protein found in the mitochondrial respiratory chain as well as the electron transfer chains of many respiratory and photosynthetic bacteria. Experiments on both mitochondrial and bacterial cyatochrome bc (1) have provided detailed kinetic information supporting a Q-cycle mechanism for electron transfer within the complex. On the basis of X-ray crystallographic studies of cytochrome bc (1), it has been proposed that the Rieske iron-sulfur protein undergoes large conformational changes as it transports electrons from ubiquinol to cytochrome c (1). A new method was developed to study electron transfer within cytochrome bc (1) using a binuclear ruthenium complex to rapidly photooxidize cytochrome c (1). The rate constant for electron transfer from the iron-sulfur center to cytochrome c (1) was found to be 80,000 s(-1), and is controlled by the dynamics of conformational changes in the iron-sulfur protein. Moreover, a linkage between the conformation of the ubiquinol binding site and the conformational dynamics of the iron-sulfur protein has been discovered which could play a role in the bifurcated oxidation of ubiquinol. A ruthenium photoexcitation method has also been developed to measure electron transfer from cytochrome c (1) to cytochrome c. The kinetics of electron transfer are interpreted in light of a new X-ray crystal structure for the complex between cytochrome bc (1) and cytochrome c.     

Characterization of the bifunctional mitochondrial processing peptidase (MPP)/bc 1 complex inSpinacia oleracea

The mitochondrial general processing peptidase (MPP) in plant mitochondria constitutes an integral part of the cytochromebc ₁ complex of the respiratory chain. Here we present a characterization of this bifunctional complex from spinach leaf mitochondria. The purified MPP/bc ₁ complex has a molecular mass of 550 kDa, which corresponds to a dimer. Increased ionic strength results in partial dissociation of the dimer as well as loss of the processing activity. Micellar concentrations of nonionic and zwitterionic detergents stimulate the activity by decreasing the temperature optimum of the processing reaction, whereas anionic detergents totally suppress the activity. MPP is a metalloendopeptidase. Interestingly, hemin, a potent regulator of mitochondrial and cytosolic biogenesis and inhibitor of proteosomal degradation, inhibits the processing activity. Measurements of the processing activity at different redox states of thebc ₁ complex show that despite bifunctionality of the MPP/bc ₁ complex, there is no correlation between electron transfer and protein processing.     

Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4

Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F₀-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture.     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.     

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     

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 stator complex of the A1A0-ATP synthase--structural characterization of the E and H subunits

Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.