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.     

Analysis of the Global Architecture of Hemoglobin A2 by Heme Binding Studies and Molecular Modeling

The kinetics of CNProto- and CNDeutero-hemin binding to apohemoglobin A₂ was investigated in a stopped-flow device in 0.05 M potassium phosphate buffer, pH 7, at 10°C. The overall kinetic profile exhibited multiple phases: Phases I–IV corresponding with heme insertion (8.5–13 × 10⁷ M^–1 s^–1), local structural rearrangement (0.21–0.23 s^–1), global structural event (0.071–0.098 s^–1), and formation of the Fe–His bond (0.009–0.012 s^–1), respectively. Kinetic differences observed between apohemoglobin A₂ and apohemoglobin A (previously studied) prompted an analysis of the structures of and chains through molecular modeling. This revealed a structural repositioning of the residues not only at, but also distant from the site of the amino acid substitutions, specifically those involved in the heme contact and subunit interface. A significant global change was observed in the structure of the exon-coded 3 region and provided additional evidence for the designation of this as the subunit assembly domain.     

Primary Steps in the Energy Conversion Reaction of theCytochrome bc 1 Complex QO Site

The primary energy conversion (Q_O) site of the cytochrome bc ₁ complex is flanked by bothhigh- and low-potential redox cofactors, the [2Fe–2S] cluster and cytochrome b _L, 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 Q_O site occupants, we have proposeda double-occupancy model for the Q_O site by ubiquinone in Rhodobacter capsulatus membranevesicles containing the cytochrome bc ₁ complex. Biophysical and biochemical experimentshave confirmed the double occupancy model and from a combination of these results and theavailable cytochrome bc ₁ crystal structures we suggest that the two ubiquinone molecules inthe Q_O site serve distinct catalytic roles. We propose that the strongly bound ubiquinone,termed Q_OS, is close to the [2Fe–2S] cluster, where it remains tightly associated with the Q_Osite during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Q_OW,is distal to the [2Fe–2S] cluster and can exchange with the membrane Q_pool on a time scalemuch faster than the turnover, acting as the substrate. The crystallographic data demonstratesthat the FeS subunit can adopt different positions. Our own observations show that theequilibrium position of the reduced FeS subunit is proximal to the Q_O site. On the basis of this, wealso report preliminary results modeling the electron transfer reactions that can occur in thecytochrome bc ₁ complex and show that because of the strong distance dependence of electrontransfer, significant movement of the FeS subunit must occur in order for the complex to beable to turn over at the experimental observed rates.     

Generation,characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

Cytochrome bc₁ complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c₂) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc₁ complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c₁ with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c₁-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5 Å resolution.     

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.     

Supramolecular organization of the photosynthetic chain in mutants of Rhodobacter capsulatus deleted in cytochrome c 2

Both the soluble cytochrome c₂ and the membrane-bound cytochrome c_y act as secondary electron carriers in photoinduced cyclic electron transfer chain of Rhodobacter capsulatus [Jenney and Daldal (1993) EMBO J 12: 1283–1292]. In this work, we have studied the kinetics of electron transfer between these secondary electron donors and the reaction center in intact cells of two mutants, MT-G4/S4 and MT-GS18 deleted in cytochrome c₂ and in cytochrome c₂ plus cytochrome bc₁ complex, respectively. In the MT-G4/S4 mutant, only about one third of the primary electron donor is reduced by cytochrome c_y in less than five ms. The remaining fraction is reduced in several seconds, although about 90% of the photoxidized cytochrome c_y is reduced in less than 10 ms by the cytochrome bc₁ complex. This implies that cytochrome c_y is not in thermodynamic equilibrium with the large fraction of primary donors which are slowly reduced. As shown by energy transfer measurements, the reaction centers connected to cytochrome c_y and the disconnected reaction centers are localized in the same membrane region. We propose that the movement of cyt c_y is restricted to a small membrane domain which includes a single cytochrome bc₁ complex. The kinetics of cytochrome c_y photooxidation in the MT-G4/S4 mutant in the presence of myxothiazol presents a fast phase (t^1/2 3 µs) followed by a slower phase (t^1/2 20 µs). In the case of the double mutant MT-GS18, the kinetics of electron transfer between cytochrome c_y and the reaction center is highly multiphasic and much slower than those observed for the MT-G4/S4 mutant. In particular, the amplitude of the fast phase is decreased by more than a factor 2 and the 20-µs phase is not observed. This implies an important structural role of the cytochrome bc₁ complex in the interaction between reaction center and cytochrome c_y, and their formation in supercomplex. The more problable stoichiometry of electron carriers in this supercomplex is 2 reaction centers, 2 cytochrome c_y and 1 cytochrome bc₁ complex.     

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     

Free energy dependence of the direct charge recombination from the primary and secondary quinones in reaction centers from Rhodobacter sphaeroides

The direct charge recombination rates from the primary quinone, k _AD (D⁺Q _A ^– DQ_A) and the secondary quinone, k _BD (D⁺Q _B ^– DQ_B), in reaction centers from Rhodobacter sphaeroides were measured as a function of the free energy differences for the processes, G _AD ⁰ and G _BD ⁰ , respectively. Measurements were performed at 21 °C on a series of mutant reaction centers that have a wide range of dimer midpoint potentials and consequently a large variation in G _AD ⁰ and G _BD ⁰ . As –G _AD ⁰ varied from 0.43 to 0.78 eV, k _AD varied from 4.6 to 28.6 s^–1. The corresponding values for the wild type are 0.52 eV and 8.9 s^–1. Observation of the direct charge recombination rate k _BD was achieved by substitution of the primary quinone with naphthoquinones in samples in which ubiquinone was present at the secondary quinone site, resulting specifically in an increase in the free energy of the D⁺Q _A ^– state relative to the D⁺Q_AQ _B ^– state. As –G _BD ⁰ varied from 0.37 to 0.67 eV, k _BD varied from 0.03 to 1.4 s^–1. The corresponding values for the wild type are 0.46 eV and 0.2 s^–1. A fit of the two sets of data to the Marcus theory for electron transfer yielded significantly different reorganization energies of 0.82 and 1.3 eV for k _AD and k _BD, respectively. In contrast, the fitted values for the coupling matrix element, or equivalently the maximum possible rate, were comparable (25 s^–1) for the two charge recombination processes. These results are in accord with Q_B having more interactions with dipoles, from both the surrounding protein and bound water molecules, than Q_A and with the primary determinant of the maximal rate being the quinone-donor distance.     

The pet operon,encoding the prosthetic group-containing subunits of the cytochrome bc1 complex,of the purple sulfur bacterium Chromatium vinosum

The pet operon, encoding the prosthetic group-containing subunits of the cytochrome bc ₁ complex of the purple sulfur bacterium Chromatium vinosum, has been cloned and sequenced. The 5 to 3 order of the C. vinosum genes is: petA, encoding the Rieske iron-sulfur protein; petB, encoding cytochrome b; and petC, encoding cytochrome c₁. Cytochrome b is the best conserved subunit of the C. vinosum complex, when compared to the corresponding proteins from four photosynthetic purple non-sulfur bacteria (70 to 74% identity). Identities for the C. vinosum Rieske protein and those from purple non-sulfur bacteria range from 60 to 64%. The C-terminal region of the C. vinosum Rieske protein is quite similar to those of purple non-sulfur bacteria, while the N-terminal region is more closely related to mitochondrial Rieske proteins of organisms such as Neurospora crassa. Cytochrome c₁ is the least well-conserved protein of the C. vinosum cytochrome bc₁ complex, with identities ranging from 49 to 51% when compared to the corresponding proteins from purple non-sulfur bacteria. A well-conserved negatively-charged region of the cytochromes c₁ of the purple non-sulfur bacteria, thought to be involved in binding the electron acceptor for the complex, cytochrome c₂, is absent in C. vinosum cytochrome c₁. A positive Southern hybridization using a probe constructed from the Rhodobacter sphaeroides fbcQ gene, which codes for a fourth subunit of the cytochrome bc₁ complex in that bacterium, suggests the presence of a homologous gene in C. vinosum.     

The Role of Various Domains of the Iron–Sulfur Protein in the Assembly and Activity of the Cytochromme bc 1 Complex of Yeast Mitochondria

Assembly studies in vitro of deletion mutants of the iron–sulfur protein into the cytochromebc ₁ complex revealed that mutants localized in the extramembranous regions of the proteinwere not assembled into the complex in contrast to the efficient assembly of mutants in themembrane-spanning region. Charged amino acids located in the extramembranous 1-4 loopand the 1 helix were mutated and expressed in yeast cells lacking the gene for the iron–sulfurprotein. Mutating the charged amino acid residues H124, E125, R146, K148, and D149 aswell as V132 and W152 resulted in loss of enzymatic activity due to the loss of iron–sulfurprotein suggesting that these amino acids are required to maintain protein stability. By contrast,no loss of iron–sulfur protein accompanied the 30–50% loss of bc ₁ complex activity in mutantsof three conserved alanine residues, A86, A90, and A92, suggesting that these residues maybe involved in the proposed movement of the flexible tether of the iron–sulfur proteinduring catalysis.     

Exogenous Ubiquinol Analogues Affect the Fluorescence of NCD-4 Bound to Aspartate-160 of Yeast Cytochrome b

Previously, we reported that the carboxyl-reacting reagent DCCD, and its fluorescent derivative NCD-4 binds covalently to aspartate-160 localized in amphipathic helix cd of the CD loop connecting membrane-spanning helices C and D of cytochrome b (Wang et al., 1995). We have investigated the fluorescent properties of NCD-4 to probe possible changes in the cd helix resulting from the binding of exogenous ubiquinol analogues to the bc ₁ complex. Preincubation of the bc ₁ complex with the reduced substrate analogues, DQH₂, DBH₂, and Q₆H₂ resulted in 20–40% increase in the fluorescence emission intensity of NCD-4 and a 10–20% increase in the binding of [¹⁴C]DCCD to the bc ₁ complex. By contrast, preincubation with the oxidized analogues DQ, DB, and Q₆ resulted in a 20–40% decrease in the fluorescence emission intensity of NCD-4 and a 20–40% decrease in the binding of [¹⁴C]DCCD to the bc ₁ complex. Moreover, addition of the reduced ubiquinols to the bc ₁ complex preincubated with NCD-4 resulted in a blue shift in the fluorescence emission spectrum. In addition, incubation of the cytochrome bc ₁ complex reconstituted into proteoliposomes with both reduced and oxidized ubiquinol analogues resulted in changes in the quenching of NCD-4 fluorescence by CAT-16, the spin-label probe that intercalates at the membrane surface. These results indicate that the addition of exogenous ubiquinol to the bc ₁ complex may result in changes in the cd helix leading to a more hydrophobic environment surrounding the NCD-4 binding site. By contrast, preincubation with the inhibitors of electron transfer through the bc ₁ complex had no effect on the binding of NCD-4 to the bc ₁ complex or on the fluorescent emission spectra, which suggests that the binding of the inhibitors does not result in changes in the environment of the NCD-4 binding site.     

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.     

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.     

Redox-linked protonation state changes in cytochrome bc1 identified by Poisson-Boltzmann electrostatics calculations

Cytochrome bc₁ is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson-Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc₁. Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc₁ from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Q_o-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron-sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Q_o-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Q_i-site) is supported by our results. A modified path for proton uptake towards the Q_i-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c₁ subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Q_i-site stabilises protons in this cluster of lysine residues.     

Genetic Evidence for Coenzyme Q Requirement in Plasma Membrane Electron Transport

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q₆ and contained greatly diminished levels of NADH–ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q₆ in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q₆ and NADH–ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3 strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3 strain grown in the presence of coenzyme Q₆. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3–14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3 strain. Ascorbate free radical reductase was not altered significantly in mutants atp2 and cor1 which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3 mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH–ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.     

Subunit 8 of theSaccharomyces cerevisiae cytochromebc 1 complex interacts with succinate-ubiquinone reductase complex

We have investigated the function of subunit 8 of the cytochromebc ₁ complex by generating six site-directed mutants, F46C, R51S, P62V, G64A, R91N, and W69-stop, in the clonedQCR8 gene and expressing the mutated genes in aSaccharomyces cerevisiae strain in which the chromosomal copy ofQCR8 is deleted. The W69-stop mutation impairs assembly of thebc ₁ complex and growth of yeast on nonfermentable carbon sources as does deletion ofQCR8 [Maarse, A. C., De Haan, M., Schoppink, P. J., Berden, J. A., and Grivell, L. A. (1988).Eur. J. Biochem. 172, 179–184], implying that the C-terminus of subunit 8 is important for assembly and/or the stability of thebc ₁ complex. The F46C, R51S, P62V, G64A, and R91N mutations do not affect the growth of yeast on nonfermentable carbon sources, not do they lower the activity or alter the inhibitor sensitivity of thebc ₁ complex. Rather, some of the mutations increase the cytochromec reductase activity of thebc ₁ complex by as much as 40%. However, succinate-ubiquinone reductase activity was consistently reduced 40–60% in mitochondrial membranes from these mutants, while NADH-ubiquinone reductase activity was not affected. In addition, the activation of succinate-ubiquinone reductase activity by succinate was diminished by the F46C, R51S, P62V, and G64A mutations. These results indicate that the cytochromebc ₁ complex participates in electron transfer from succinate to ubiquinonein situ and also suggest an interaction between succinate-ubiquinone reductase and cytochromebc ₁ complex which involves subunit 8 of thebc ₁ complex.     

Electron donation from membrane-bound cytochrome c to the photosynthetic reaction center in whole cells and isolated membranes of Heliobacterium gestii

The reaction between membrane-bound cytochrome c and the reaction center bacteriochlorophyll g dimer P798 was studied in the whole cells and isolated membranes of Heliobacterium gestii. In the whole cells, the flash-oxidized P798⁺ was rereduced in multiple exponential phases with half times (t _1/2s) of 10 s, 300 s and 4 ms in relative amplitudes of 40, 35 and 25%, respectively. The faster two phases were in parallel with the oxidation of cytochrome c. In isolated membranes, a significantly slow oxidation of the membrane-bound cytochrome c was detected with t _1/2 = 3 ms. This slow rate, however, again became faster with the addition of Mg²⁺. The rate showed a high temperature dependency giving apparent activation energies of 88.2 and 58.9 kJ/mol in the whole cells and isolated membranes, respectively. Therefore, membrane-bound cytochrome c donates electrons to the P798⁺ in a collisional reaction mode like the reaction of water-soluble proteins. The rereduction of the oxidized cytochrome c was suppressed by the addition of stigmatellin both in the whole cells and isolated membranes. This indicates that the electron transfer from the cytochrome bc complex to the photooxidized P798⁺ is mediated by the membrane-bound cytochrome c. The multiple flash excitation study showed that 2–3 hemes c were connected to the P798. By the heme staining after the SDS-PAGE analysis of the membraneous proteins, two cytochromes c were detected on the gel indicating apparent molecular masses of 17 and 30 kDa, respectively. The situation resembles the case in green sulfur bacteria, that is, the membrane-bound cyotochrome c _z couples electron transfer between the cytochrome bc complex and the P840 reaction center complex.This revised version was published online in October 2005 with corrections to the Cover Date.     

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Cytochrome bc₁ isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc₁ is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc₁ was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc₁ retained nearly full activity, absorption spectra, subunit, and phospholipid composition.     

Conformational Change of the Rieske [2Fe–2S] Protein inCytochrome bc 1 Complex

Structures of mitochondrial bc ₁ complex have been reported based on four different crystalforms by three different groups. In these structures, the extrinsic domain of the Rieske [2Fe–2S]protein, surprisingly, appeared at three different positions: the c ₁ position, where the [2Fe–2S]cluster exists in close proximity to the heme c ₁; the b position, where the [2Fe–2S] clusterexist in close proximity to the cytochrome b; and the intermediate position where the[2Fe–2S] cluster exists in between c ₁ and b positions. The conformational changes betweenthese three positions can be explained by a combination of two rotations; (1) a rotation of theentire extrinsic domain and (2) a relative rotation between the cluster-binding fold and thebase fold within the extrinsic domain. The hydroquinone oxidation and the electron bifurcationmechanism at the Q_P binding pocket of the bc ₁ complex is well explained using theseconformational changes of the Rieske [2Fe–2S] protein.     

An isolated reaction center complex from the green sulfur bacterium Chlorobium vibrioforme can photoreduce ferredoxin at high rates

Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617–9624). In the presence of ferredoxin and ferredoxin:NADP⁺ oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP⁺ at rates of 333 and 110 mol (mg bacteriochlorophyll a)^–1 h^–1, respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c ₅₅₁ and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c ₅₅₁ inhibit NADP⁺ reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.Abbreviations FMO protein Fenna-Matthews-Olson protein - TMBZ 3,3,5,5-tetramethylbenzidine