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Photochemical and ligand-exchange properties of the cyanide complex of fully reduced cytochrome c oxidase.

ABSTRACT: Cytochrome oxidase, in its fully reduced state, forms a complex with CN having a Kd of 230 microM with a stoicheiometry of 1 CN molecule per cytochrome oxidase. We do not detect a second CN-binding site as seen by i.r. spectroscopy [Yoshikawa & Caughey (1990) J. Biol. Chem. 265, 7945-7958]. The ferrocytochrome a3-CN complex, like the analogous ferrocytochrome a3-CO complex, is photosensitive but with a 15-fold lower quantum yield for photolysis. Analysis of the recombination kinetics after CN photolysis establishes a simple bimolecular binding constant of 235 M-1.s-1, in agreement with the value obtained from stopped-flow studies [Antonini, Brunori, Greenwood, Malmström & Rotillo (1971) Eur. J. Biochem. 23, 396-400]. A rate of 0.07 s-1 for the first-order dissociation of CN from cytochrome a3 is found by the rate of exchange of CO with ferrocytochrome a3-CN, and is consistent with the value calculated from the equilibrium binding constant and the CN on rate. However, O2 is able to oxidize the fully reduced CN compound at a rate well in excess of the CN off rate. The product of this oxidation reaction is a partially reduced CN complex. This implies that O2 either promotes CN dissociation or is able to oxidize the CN-bound enzyme directly. These results are discussed in the context of the structure and dynamics of the ligand-binding site of cytochrome oxidase.

SUBMITTER: Hill BC

PROVIDER: S-EPMC1151613 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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Project description:Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.

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Photochemical and ligand-exchange properties of the cyanide complex of fully reduced cytochrome c oxidase.

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