Effect of calcium ions on electron transfer between hemes a and a(3) in cytochrome c oxidase.

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH(3))(6)(3+), three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a(3) reduction. The difference spectrum of heme a(3) reduction in the visible region is characterized by a maximum at ~612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a(3) reduction is slowed down by low concentrations of Ca(2+). The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca(2+) ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca(2+) ions discovered earlier and indicate that the cation affects intramolecular electron transfer.