Kinetic studies on the binding of cyanide to oxygenated cytochrome c oxidase.
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ABSTRACT:
SUBMITTER: Brittain T
PROVIDER: S-EPMC1172854 | biostudies-other | 1976 May
REPOSITORIES: biostudies-other
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Project description:Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P?F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F?O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P?F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F?O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.
| S-EPMC3139697 | biostudies-literature
Project description: Not available
| S-EPMC8488603 | biostudies-literature
Project description:The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be 'pulsed', i.e. activated, like the mammalian enzyme. The 'pulsed' species is spectroscopically different from the 'resting' one, and the decay into the 'resting' state is fast (t1/2 approx. 3 min).
| S-EPMC1152413 | biostudies-other
Project description: Not available
| S-EPMC8826895 | biostudies-literature
Project description:Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs.
| S-EPMC2728958 | biostudies-literature
Project description:Pseudomonas pseudoalcaligenes CECT5344 grows in minimal medium containing cyanide as the sole nitrogen source. Under these conditions, an O2-dependent respiration highly resistant to cyanide was detected in cell extracts. The structural genes for the cyanide-resistant terminal oxidase, cioA and cioB, are clustered and encode the integral membrane proteins that correspond to subunits I and II of classical cytochrome bd, although the presence of heme d in the membrane could not be detected by difference spectra. The cio operon from P. pseudoalcaligenes presents a singular organization, starting upstream of cioAB by the coding sequence of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. PCR amplifications of RNA (reverse transcription-PCR) indicated the cyanide-dependent up-regulation and cotranscription along the operon. The targeted disruption of cioA eliminates both the expression of the cyanide-stimulated respiratory activity and the growth with cyanide as the nitrogen source, which suggests a critical role of this cytochrome bd-related oxidase in the metabolism of cyanide by P. pseudoalcaligenes CECT5344.
| S-EPMC1950984 | biostudies-literature
Project description:In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.
| S-EPMC1165384 | biostudies-other
Project description:We examined the anion binding behavior of the Mg(Mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. Rhodobacter sphaeroides grown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. Due to its close proximity and a shared ligand, oxidized Cu(A) is spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine and R.s. oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when Cu(A) is oxidized. In the reduced structure, one water molecule in the vicinity of the Cu(A) ligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochrome c oxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.
| S-EPMC2638054 | biostudies-literature
Project description:Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32±0.16 M(-1) s(-1) and 0.34±0.15 s(-1), respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1±0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a "peroxygenase"-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min(-1) at pH 6.0.
| S-EPMC3896955 | biostudies-literature
Project description:Cytochrome c oxidase from ox heart was inserted into artificial liposomal vesicles obtained by sonication of purified soya-bean phospholipids. The cytochrome oxidase vesicles showed a respiratory control ratio of about 2. Spectroscopic properties in the visible and Soret regions and kinetics of CO binding are similar to those of the soluble oxidase. The catalytic efficiency of the cytochrome oxidase vesicles in oxidizing cytochrome c increases as a result of the formation of the 'pulsed' form of the oxidase and of the presence in the reaction mixture of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and nonactin. Analysis of the experimental results obtained under several conditions supports the conclusions that: (i) the alkalinization of the internal microenvironment in the liposomal vesicle is not by itself responsible for the decrease in catalytic activity; (ii) the electrical potential difference created during turnover by proton consumption and/or pumping through the liposome wall is an important mechanism of control in the chain of events leading to the oxidation of external cytochrome c.
| S-EPMC1154058 | biostudies-other