Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking.
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.
Malatesta
F
F
Dipartimento di Biologia di Base ed Applicata, Universita' di L'Aquila, Italy.
Antonini
G
G
Nicoletti
F
F
Giuffrè
A
A
D'Itri
E
E
Sarti
P
P
Brunori
M
M
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000737:chemistry; D045304:Cytochromes c; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins
C014059:CYC1 protein, S cerevisiae; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D003576:Electron Transport Complex IV