| Abstract: | We report studies in which we have used N-(2,2,6,6-tetramethylpiperidyl-1-oxyl)-N′-cyclohexylcarbodiimide, a spinlabel analogue of N,N′-dicyclohexylcarbodiimide, to investigate the structural aspects of the cytochrome c oxidase proton pump. We establish that the spin label binds to the reconstituted enzyme at the same site as does N,N′-dicyclohexylcarbodiimide, i.e., within subunit III. ESR studies of the bound spin label indicate that its binding site is situated in an apolar region of the enzyme, though close to its surface. The binding of the spin label to the free oxidase is different from that with the reconsituted enzyme, leading to spin-spin exchange between the bound probe molecules. From this and the fact that N,N′-dicyclohexylcarbodiimide binds to subunits III and IV in the free oxidase, we conclude that these two subunits are at the most 20 Å apart. |
