The oxygen reactive species of cytochrome-c-oxidase: an alternative view.
In a recent review article Babcok and Wikström (Nature, 1992, 356, 301-309) proposed that the species of cytochrome-c-oxidase which binds molecular oxygen during turnover is the so-called mixed valence enzyme, in which the binuclear center cytochrome a3-CuB is reduced, while the cytochrome a/CuA sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948-958) in which it was found that the steady-state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady-state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady-state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid-scanning stopped-flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome-c-oxidase as defined above is not the prevailing oxygen binding species of cytochrome-c-oxidase, unless electron donation to cytochrome c becomes rate limiting.
Brunori
M
M
Department of Biochemical Sciences, University of Rome, La Sapienza, Italy.
Antonini
G
G
Malatesta
F
F
Sarti
P
P
Wilson
M T
MT
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D003576:Electron Transport Complex IV; D013772:Tetramethylphenylenediamine; D001205:Ascorbic Acid; D010100:Oxygen