Freedman J A, Dyer B, Tattrie B, Nicholls P
Syracuse Research Corporation, NY 13244.
Biochim Biophys Acta. 1993 Jul 10;1164(2):138-42. doi: 10.1016/0167-4838(93)90240-r.
Binding of antibodies raised against subunit V of mammalian cytochrome oxidase to the intact membranous enzyme is redox-sensitive, suggesting the existence of 'open' and 'closed' protein conformers (Freedman, J.A., Cooper, C.E., Leece, B., Nicholls, P. and Chan, S.H.P. (1988) Biochem. Cell Biol. 66, 1210-1217). Similar open and closed states for the oxygen-reacting site have been proposed to explain cyanide binding kinetics (Jensen, P., Wilson, M.T., Aasa, R. and Malmström, B.G. (1984) Biochem. J. 224, 829-837). We therefore examined cyanide inhibition of oxidase activity polarographically and spectrophotometrically using soluble oxidase preincubated with and without anti-subunit V or non-immune rabbit gamma-globulin. The subunit-specific antibody decreased the cyanide 'on' rate and essentially eliminated the rapid phase of cyanide binding. We conclude that (i), bound antibody blocks HCN binding; (ii), antibody and HCN probably bind to the same conformation of the oxidase and (iii), the 'open'-'closed' conformation change that modulates binding of HCN may be similar to that which modulates antibody binding. The results are consistent with some reciprocating models of electron transfer and energy transduction by the oxidase (cf., Wikström, M.K.F., Krab, K. and Saraste, M. (1981) Cytochrome Oxidase: A Synthesis).
针对哺乳动物细胞色素氧化酶亚基V产生的抗体与完整的膜状酶的结合对氧化还原敏感，这表明存在“开放”和“封闭”的蛋白质构象（弗里德曼，J.A.，库珀，C.E.，利西，B.，尼科尔斯，P.和陈，S.H.P.（1988年）《生物化学与细胞生物学》66卷，1210 - 1217页）。有人提出氧反应位点类似的开放和封闭状态来解释氰化物结合动力学（詹森，P.，威尔逊，M.T.，阿萨，R.和马尔姆斯特伦，B.G.（1984年）《生物化学杂志》224卷，829 - 837页）。因此，我们使用预先与抗亚基V或非免疫兔γ球蛋白一起孵育或未孵育的可溶性氧化酶，通过极谱法和分光光度法研究了氰化物对氧化酶活性的抑制作用。亚基特异性抗体降低了氰化物的“结合”速率，并基本上消除了氰化物结合的快速阶段。我们得出结论：（i），结合的抗体阻断HCN结合；（ii），抗体和HCN可能结合到氧化酶的相同构象；（iii），调节HCN结合的“开放” - “封闭”构象变化可能与调节抗体结合的构象变化相似。这些结果与氧化酶的一些电子传递和能量转导的往复模型一致（参见，维克斯特伦，M.K.F.，克拉布，K.和萨拉斯特，M.（1981年）《细胞色素氧化酶：综述》）。
