* To whom correspondence should be addressed.
Received February 28, 2012; Revision received March 15, 2012
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(NH3)63+, three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a3 reduction. The difference spectrum of heme a3 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 a3 reduction is slowed down by low concentrations of Ca2+. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca2+ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca2+ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.
KEY WORDS: cytochrome c oxidase, Ca2+ ions, fast kinetics, heme a3