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Probing the oxygen binding site of cytochrome c oxidase with cyanide
OXYGEN
309
DO42
PROBING THE OXYGENBINDING SITEOF CYTOCHRONE C OXIDASE WITH CYANIDE N.T. Wilson4 University oft Essex, Ut+ited Kingdom, 6. Antonini , F. Nalatesta P. Sarti and N. Brunori* Universities “Tar Vergata”*, “La Sapienzo”s and CNR*, Roma,
Italy.
Cyanide binds rapidly and with high affinity only to partially reduced forms of cytochrome c oxidase populated during 'turnover'[l]. The number of electrons cytochrome 2 oxidase must accept before cyanide can rapidly bind and the location of these electrons (or electron) within the set of redox centres available (Cu,, haem 3, Cug, haem 3s) remains controversial. It is these questions we have recently attempted to answer by application of 'double mixing' and 'rapid scan' stopped-flow spectrophotometry. Rapid scan kinetic experiments have confirmed earlier findings that inhibition requires prior electron entry into the enzyme and that the kinetics of inhibition are strongly cyanide concentration dependent. There is no influence of oxygen concentration, however,, demonstratinq that oxygen and cyanide do not compete for the same site. We have shown that full inhibition of the enzyme can occur within 50 ms, a time comparable with the initial electron entry 'burst' into the enzyme. The complex optical spectra have been analysed by Singular Value Decomposition (SVD) allowing deconvolution of the spectra. These analytical procedures show that entry of a single (1 - 1.3) electron from cytochrome c is sufficient to permit rapid cyanide binding. This electron, once cyanide has bound, is located on the Cu,/haem 2 centre. The nature of the spectroscopic changes induced by rapid and inhibitory cyanide binding seem to exclude cytochrome 3s as the primary binding site. 2w+e conclude therefore that cyanide binds initially to Cu and that access to this site is effected by entry of a singale electron into the enzyme. Our conclusion that CuB2' is the primary cyanide acceptor is in line with experiments which suggest that this metal is on the pathway which extrinsic ligands must follow from the exterior to haem as [e.g. 21. Both oxygen and CO also bind initially to this site but in this case to the Cu,* state consistent with the lack of competition between gyanide and trapping oxygen in our experiments. Cyanide binding to Cu this metal in the cupric state may thus in$libit rapid electron transfer to both Cu, and haem 3s.
1. 2.
M.G. Jones,
D. Bickar, M.T. Wilson, M. Brunori, A.Colosimo and P.Sarti, BiochemJ., 220, 57 (1984). Blackmore, R.S., Greenwood, C., and Gibson, Q.H., J. BioZ. Chem. , 266, 19245 (1991) .
309
DO42
PROBING THE OXYGENBINDING SITEOF CYTOCHRONE C OXIDASE WITH CYANIDE N.T. Wilson4 University oft Essex, Ut+ited Kingdom, 6. Antonini , F. Nalatesta P. Sarti and N. Brunori* Universities “Tar Vergata”*, “La Sapienzo”s and CNR*, Roma,
Italy.
Cyanide binds rapidly and with high affinity only to partially reduced forms of cytochrome c oxidase populated during 'turnover'[l]. The number of electrons cytochrome 2 oxidase must accept before cyanide can rapidly bind and the location of these electrons (or electron) within the set of redox centres available (Cu,, haem 3, Cug, haem 3s) remains controversial. It is these questions we have recently attempted to answer by application of 'double mixing' and 'rapid scan' stopped-flow spectrophotometry. Rapid scan kinetic experiments have confirmed earlier findings that inhibition requires prior electron entry into the enzyme and that the kinetics of inhibition are strongly cyanide concentration dependent. There is no influence of oxygen concentration, however,, demonstratinq that oxygen and cyanide do not compete for the same site. We have shown that full inhibition of the enzyme can occur within 50 ms, a time comparable with the initial electron entry 'burst' into the enzyme. The complex optical spectra have been analysed by Singular Value Decomposition (SVD) allowing deconvolution of the spectra. These analytical procedures show that entry of a single (1 - 1.3) electron from cytochrome c is sufficient to permit rapid cyanide binding. This electron, once cyanide has bound, is located on the Cu,/haem 2 centre. The nature of the spectroscopic changes induced by rapid and inhibitory cyanide binding seem to exclude cytochrome 3s as the primary binding site. 2w+e conclude therefore that cyanide binds initially to Cu and that access to this site is effected by entry of a singale electron into the enzyme. Our conclusion that CuB2' is the primary cyanide acceptor is in line with experiments which suggest that this metal is on the pathway which extrinsic ligands must follow from the exterior to haem as [e.g. 21. Both oxygen and CO also bind initially to this site but in this case to the Cu,* state consistent with the lack of competition between gyanide and trapping oxygen in our experiments. Cyanide binding to Cu this metal in the cupric state may thus in$libit rapid electron transfer to both Cu, and haem 3s.
1. 2.
M.G. Jones,
D. Bickar, M.T. Wilson, M. Brunori, A.Colosimo and P.Sarti, BiochemJ., 220, 57 (1984). Blackmore, R.S., Greenwood, C., and Gibson, Q.H., J. BioZ. Chem. , 266, 19245 (1991) .