Watmough N J, Katsonouri A, Little R H, Osborne J P, Furlong-Nickels E, Gennis R B, Brittain T, Greenwood C
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norfolk, United Kingdom.
Biochemistry. 1997 Nov 4;36(44):13736-42. doi: 10.1021/bi971434i.
We have compared the reactions with dioxygen of wild-type cytochrome bo3 and a mutant in which a conserved glutamic acid at position-286 of subunit I has been changed to an alanine. Flow-flash experiments reveal that oxygen binding and the rate of heme-heme electron transfer are unaffected by the mutation. Reaction of the fully (3-electron) reduced mutant cytochrome bo3 with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen [Puustinen, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1545-1548]. These results confirm that proton uptake is important in controlling the later stages of dioxygen reduction in heme-copper oxidases and show that E286 is an important component of the channel that delivers these protons to the active site.
我们比较了野生型细胞色素bo3与一种突变体和双氧的反应，该突变体中I亚基第286位保守的谷氨酸被替换为丙氨酸。流动闪光实验表明，氧结合以及血红素 - 血红素电子转移速率不受该突变影响。完全（三电子）还原的突变体细胞色素bo3与双氧反应产生一个双核中心，该中心基本上处于P（过氧）状态，而非完全还原的野生型酶与双氧反应产物中特征明确的F（氧铁）状态[普斯蒂宁，A.等人（1996年）《美国国家科学院院刊》93，1545 - 1548]。这些结果证实质子摄取在控制血红素 - 铜氧化酶中双氧还原后期阶段很重要，并表明E286是将这些质子传递到活性位点的通道的重要组成部分。
