Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: crosslinking in the high- and low-affinity binding sites

Abstract

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the 2nd modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c cross-linked both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence also suggests that cytochrome c bound to the lipid was in the low-affinity binding site. Covalent binding of the cytochrome c derivative into the high-affinity binding site inhibited electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was convalently bound to the enzyme per cytochrome c oxidase dimer (i.e., .simeq. 280,000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.