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. 1996 May 1;315(Pt 3):909–916. doi: 10.1042/bj3150909

Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking.

F Malatesta 1, G Antonini 1, F Nicoletti 1, A Giuffrè 1, E D'Itri 1, P Sarti 1, M Brunori 1
PMCID: PMC1217293  PMID: 8645176

Abstract

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046-7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme. However, (3) K(m) and Vmax values obtained from the non-linear Eadie-Hofstee plots are very similar in both caa3 and aa3. The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

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Selected References

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