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1.
Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec 552, and thea-type terminal oxidase. Deletion mutants in thebc 1 and theaa 3 complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec 552 in electron transport between complex III and IV is presented.  相似文献   

2.
Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [35S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec 558 and has monophasic kinetics. Using horse cytochromec 550 as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec 550 and 35-fold higher with theEuglena cytochromec 558. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number  相似文献   

3.
The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O2 concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O2 concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O2 concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.  相似文献   

4.
Summary The interference of cytochromec with absorption and fluorescence changes of the cyanine dye, diS-C3-(5), was investigated in the presence of liposomes and cytochromec-oxidase reconstituted proteoliposomes. The apparent cytochromec-dependent quenching of diS-C3-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochromec-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochromec-induced breakdown of dye was observed in the presence of hydrogen peroxide.The cytochromec mediated absorbance and fluorescence losses of diS-C3-(5) in liposomal or proteoliposomal systems are prevented by Ca2+ and La3+ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochromec is inhibited either directly (e. g. by BHT) or indirectly, by preventing the binding of cytochromec to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential inaa 3-reconstituted proteoliposomes is discussed.  相似文献   

5.
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 (Qo) site or the quinol reductase (Qi) 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.  相似文献   

6.
Various direct, indirect (kinetic and thermodynamic), and combined mechanisms have been proposed to explain the conversion of redox energy into a transmembrane protonmotive force (p) by enzymatic complexes of respiratory chains. The conceptual evolution of these models is examined. The characteristics of thermodynamic coupling between redox transitions of electron carriers and scalar proton transfer in cytochromec oxidase and its possible involvement in proton pumping is discussed. Other aspects dealt with in this paper are: (i) variability of H+/e stoichiometries, in cytochromec oxidase and cytochromec reductase and its mechanistic implications; (ii) possible models by which the reduction of dioxygen to water at the binuclear heme-copper center of protonmotive oxidases can be directly involved in proton pumping. Finally a unifying concept for proton pumping by the redox complexes of respiratory chain is presented.  相似文献   

7.
The subject of this short review is the cytochromec oxidase (caa 3) from the thermophilic bacteriumThermus thermophilus. First, some of the extensive physical and enzymological results obtained with this enzyme are reviewed, and two experiments are described, involving isotope substitutions in combination with Mössbauer and ENDOR spectroscopies, which have provided novel insight into the active sites of the enzyme. Second, we summarize recent molecular genetic work showing thatThermus cytochromecaa 3 is abona fide member of the superfamily of heme-copper oxidases. Finally, we present a rough three-dimensional model and speculate about certain features of the metal-binding sites.  相似文献   

8.
Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to CuA, to cytochromea, and then to the binuclear (i.e., cytochromea 3-CuB) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to CuA 5–7 Å, cytochromec to cytochromea 20–25 Å, CuA to cytochromea 14–16 Å and cytochromea to cytochrome a3-CuB 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.  相似文献   

9.
Paracoccus denitrificans is able to grow on the C1 compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an 22 configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec 551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec 1 and cytochromec 552 and the periplasmic-located cytochromec 550 are present under all tested growth conditions. The cytochromesc 551i andc 553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec 1, cytochromec 550, cytochromec 551i , and cytochromec 553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C1 compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec 551i . Further electron transport is either via cytochromec 550 or cytochromec 553i to cytochromeaa 3. However, direct electron transport from cytochromec 551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec 550 to cytochromeaa 3, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.  相似文献   

10.
Cytochromeaa 3 ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea 3 (oro)-CuB center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.  相似文献   

11.
Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec 552 than with either solubleParacoccus c 550 or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.  相似文献   

12.
The interaction of reduced rabbit cytochrome b5 with reduced yeast iso-1 cytochrome c has been studied through the analysis of 1H–15N HSQC spectra, of 15N longitudinal (R1) and transverse (R2) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b5 has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b5.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics  相似文献   

13.
Cytochrome c3 from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c3 with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c3 molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c3 is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c3 to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c3 and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c3 stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c3 facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c3 donates the accepted electrons to the viologen with difficulty.  相似文献   

14.
Preparations and protein chemical characterizations performed with cytochromec oxidase (E.C. 1.9.3.1) from the purple bacteriumParacoccus denitrificans are reviewed. The simplest catalytically competent complex of the enzyme consists of two subunits of 62012 and 27999 Da. The theoretical hemea/protein ratio of the purified enzyme is 22.0 nmol/mg. The amino acid sequences of both proteins are compared with examples of subunits I and II of mitochondrial terminal oxidases from the main kingdoms of eukaryotes. The significance of the emerging conserved features such as membrane penetration patterns, invariant residues, stoichiometry, and sites of prosthetic groups are discussed. TheParacoccus enzyme represents the only prokaryotic oxidase detailed so far, which is directly related to the mitochondrial oxidases by common ancestry in the growing O2 atmosphere.  相似文献   

15.
The electronic transitions of the two heme groups of cytochromec oxidase have been resolved by application of second-derivative and cryogenic absorption spectroscopy. Both methods reveal a splitting of the ferrocytochromea Soret transition into two features at 443 and 450 nm. The relative intensity of the 450 nm feature appears to depend on the ligation state of cytochromea 3, the solution pH, and complex formation with cytochromec. The structural origin and mechanistic significance of this second Soret transition of cytochromea are discussed in terms of the electron transfer and proton translocation activities of the enzyme.Dedicated to the memory of James Carl Copeland.  相似文献   

16.
The K+-ionophores valinomycin and nonactin induce a qualitatively identical change of the visible spectrum of isolated oxidized cytochromec oxidase (red shift), but the amplitude is half with nonactin. Valinomycin, in the presence or absence of a protonophore, stimulates the respiration of the reconstituted enzyme to a higher extent than nonactin and results in a higherK m for cytochromec. In contrast, nonactin causes a fivefold rate of proton conductivity across a liposomal membrane, after induction of a K+-diffusion potential. The data indicate that respiratory control by these antibiotics is not only due to degradation of a membrane potential, but rather to specific interaction with and modification of cytochromec oxidase.  相似文献   

17.
Several loci on theParacoccus denitrificans chromosome are involved in the synthesis of cytochromec oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochromec 550 gene. An alignment of six promoter regions reveals only short common sequences.  相似文献   

18.
Cytochrome cbb 3 oxidase, a member of the heme–copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant histidine residues, which are diagnostic of heme–copper oxidases in all type of the heme–copper oxidase superfamily. Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb 3-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN subunits of the cbb 3 oxidase, but not the catalytic subunits of other members of heme–copper oxidases superfamily. The function of these six CcoN-specific conserved histidines of cbb 3-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly of the Rhodobacter capsulatus cbb 3-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared with wild-type oxidase.  相似文献   

19.
Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec 2 was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec 2 is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec 2.  相似文献   

20.
It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec 550 and cytochromec 552, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec 552 and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.  相似文献   

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