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 oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25-1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme 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 required 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 reduction of the O(2)-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original 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, consistently with a peroxide at the O(2) reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K. , Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998) Science 280, 1723-1729).
Mochizuki
M
M
Department of Life Science, Himeji Institute of Technology, CREST, Japan Science and Technology Corporation, Kamigohri Akoh, Hyogo 678-1297, Japan.
Aoyama
H
H
Shinzawa-Itoh
K
K
Usui
T
T
Tsukihara
T
T
Yoshikawa
S
S
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004227:Dithionite / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology*; D010084:Oxidation-Reduction
D004227:Dithionite; D003576:Electron Transport Complex IV