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Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation.

ABSTRACT: The inhibition of cytochrome c oxidase by cyanide, starting either with the resting or the pulsed enzyme, was studied by rapid-freeze quenching followed by quantitative e.p.r. It is found that a partial reduction of cytochrome oxidase by transfer of 2 electron equivalents from ferrocytochrome c to cytochrome a and CuA will induce a transition from a closed to an open enzyme conformation, rendering the cytochrome a3-CuB site accessible for cyanide binding, possibly as a bridging ligand. A heterogeneity in the enzyme is observed in that an e.p.r. signal from the cytochrome a3 3+-HCN complex is only found in 20% of the molecules, whereas the remaining cyanide-bound a3-CuB sites are e.p.r.-silent.

SUBMITTER: Jensen P

PROVIDER: S-EPMC1144519 | biostudies-other | 1984 Dec

REPOSITORIES: biostudies-other

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Project description:The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.

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Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation.

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