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 a 3-Cu B is reduced, while the cytochrome a/Cu A 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.
