The oxygen reactive species of cytochrome‐c‐oxidase: An alternate view

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Abstract
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.