Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase.
Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.
Nilsson
T
T
Arthur Amos Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125.
Gelles
J
J
Li
P M
PM
Chan
S I
SI
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006902:Hydroxymercuribenzoates / Q000494:pharmacology*; D007700:Kinetics; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology; D011485:Protein Binding
D003574:Cytochrome c Group; D006902:Hydroxymercuribenzoates; D008081:Liposomes; C030601:4-hydroxymercuribenzoate; D003300:Copper; D003576:Electron Transport Complex IV