Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase

Approximately 30% of the iron contained in a bovine heart cytochrome c oxid ase preparation was removed by crystallization, giving a molecular extincti on coefficient 1.25-1.4 times higher than those reported thus far. Six elec tron equivalents provided by dithionite were required for complete reductio n of the crystalline cytochrome c oxidase preparation. The fully reduced en zyme was oxidized with 4 oxidation equivalents provided by molecular oxygen , giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were r equired for complete reduction of the O-2-oxidized enzyme. The O-2-oxidized form, when exposed to excess amounts of O-2, was converted to the original oxidized form which required 6 electrons for complete reduction. A slow re duction of the O-2-oxidized form without any external reductant added indic ates the existence of internal electron donors for heme irons in the enzyme . These results suggest that the 2 extra oxidation equivalents in the origi nal oxidized form, compared with the O-2-oxidized form, are due to a bound peroxide produced by O-2 and electrons from the internal donors, consistent ly with a peroxide at the O-2 reduction site in the crystal structure of th e enzyme (Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamas hita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-172 9).