Time-resolved generation of a membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus. Evidence for reduction-induced opening of the binuclear center.
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ba3-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a microsecond/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba3 by RuBpy results in two phases of membrane potential generation by the enzyme with tau values of about 20 and 300 microseconds at pH 8 and 23 degrees C. Spectrophotometric experiments show that oxidized ba3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of approximately 2 x 10(3) M-1 s-1. The data indicate that single electron reduction of ba3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba3 oxidase as compared to the oxidases of the aa3-type. (+info)
Exercise intolerance due to mutations in the cytochrome b gene of mitochondrial DNA.
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BACKGROUND: The mitochondrial myopathies typically affect many organ systems and are associated with mutations in mitochondrial DNA (mtDNA) that are maternally inherited. However, there is also a sporadic form of mitochondrial myopathy in which exercise intolerance is the predominant symptom. We studied the biochemical and molecular characteristics of this sporadic myopathy. METHODS: We sequenced the mtDNA cytochrome b gene in blood and muscle specimens from five patients with severe exercise intolerance, lactic acidosis in the resting state (in four patients), and biochemical evidence of complex III deficiency. We compared the clinical and molecular features of these patients with those previously described in four other patients with mutations in the cytochrome b gene. RESULTS: We found a total of three different nonsense mutations (G15084A, G15168A, and G15723A), one missense mutation (G14846A), and a 24-bp deletion (from nucleotide 15498 to 15521) in the cytochrome b gene in the five patients. Each of these mutations impairs the enzymatic function of the cytochrome b protein. In these patients and those previously described, the clinical manifestations included progressive exercise intolerance, proximal limb weakness, and in some cases, attacks of myoglobinuria. There was no maternal inheritance and there were no mutations in tissues other than muscle. The absence of these findings suggests that the disorder is due to somatic mutations in myogenic stem cells after germ-layer differentiation. All the point mutations involved the substitution of adenine for guanine, but all were in different locations. CONCLUSIONS: The sporadic form of mitochondrial myopathy is associated with somatic mutations in the cytochrome b gene of mtDNA. This myopathy is one cause of the common and often elusive syndrome of exercise intolerance. (+info)
The active form of the ferric heme in neutrophil cytochrome b(558) is low-spin in the reconstituted cell-free system in the presence of amphophil.
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The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b(558) activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b(558) activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide. (+info)
Steered molecular dynamics simulation of the Rieske subunit motion in the cytochrome bc(1) complex.
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Crystallographic structures of the mitochondrial ubiquinol/cytochrome c oxidoreductase (cytochrome bc(1) complex) suggest that the mechanism of quinol oxidation by the bc(1) complex involves a substantial movement of the soluble head of the Rieske iron-sulfur protein (ISP) between reaction domains in cytochrome b and cytochrome c(1) subunits. In this paper we report the results of steered molecular dynamics simulations inducing, through an applied torque within 1 ns, a 56 degrees rotation of the soluble domain of ISP. For this purpose, a solvated structure of the bc(1) complex in a phospholipid bilayer (a total of 206,720 atoms) was constructed. A subset of 91,061 atoms was actually simulated with 45,131 moving atoms. Point charge distributions for the force field parametrization of heme groups and the Fe(2)S(2) cluster of the Rieske protein included in the simulated complex were determined. The simulations showed that rotation of the soluble domain of ISP is actually feasible. Several metastable conformations of the ISP during its rotation were identified and the interactions stabilizing the initial, final, and intermediate positions of the soluble head of the ISP domain were characterized. A pathway for proton conduction from the Q(o) site to the solvent via a water channel has been identified. (+info)
An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis.
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The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to self-splice or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent salt concentrations. This two-component system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing. (+info)
Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.
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In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed. (+info)
Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.
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Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis. (+info)
Ubiquinone at center N is responsible for triphasic reduction of cytochrome b in the cytochrome bc(1) complex.
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We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc(1) complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction. With cytochrome bc(1) complex from wild type yeast, cytochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase. Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c(1). With cytochrome bc(1) complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction by either menaquinol or ubiquinol was rapid and monophasic. Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased. When reduction of the cytochrome bc(1) complex through center P was blocked, cytochrome b reduction through center N was biphasic and was slowed by the addition of exogenous ubiquinone. These results show that ubiquinone residing at center N in the oxidized cytochrome bc(1) complex is responsible for the triphasic reduction of cytochrome b. (+info)