Munro A W, Malarkey K, McKnight J, Thomson A J, Kelly S M, Price N C, Lindsay J G, Coggins J R, Miles J S
Department of Biochemistry, University of Glasgow, U.K.
Biochem J. 1994 Oct 15;303 ( Pt 2)(Pt 2):423-8. doi: 10.1042/bj3030423.
The 'Covalent Switching' hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group.
“共价开关”假说表明，一个高度保守的色氨酸残基在细胞色素P-450系统中作为氧化还原伙伴之间电子传递流的介质[鲍德温、莫里斯和理查兹（1991年）《伦敦皇家学会学报》B辑245，43 - 51]。我们研究了巨大芽孢杆菌细胞色素P-450 BM3（P-450 102）中保守色氨酸（Trp-97）改变的影响。用丙氨酸、苯丙氨酸或酪氨酸取代Trp-97会导致天然血红素含量降低，并改变纯化突变酶中剩余血红素的静止自旋状态。然而，动力学分析表明突变酶保留了高水平的催化活性。圆二色光谱和电子顺磁共振光谱也显示二级结构几乎没有变化，血红素连接模式也没有改变。这些发现对P-450s分子间电子流的共价开关机制提出了质疑，但表明该残基在血红素辅基的结合中起作用。
