Sligar S G, Kennedy K A, Pearson D C
Proc Natl Acad Sci U S A. 1980 Mar;77(3):1240-4. doi: 10.1073/pnas.77.3.1240.
Using isotopic tracer methods, we have shown that dihydrolipoic acid (2,3-thioctic acid) acylates the distal oxygen of ferrous oxygenated Pseudomonas cytochrome P-450, forming a transient acyl peroxide intermediate that facilitates oxygen-oxygen bond cleavage. Single-turnover studies with 18O2 indicate one oxygen-18 atom incorporated into the carboxylate group of lipoic acid for each oxygen-18 inserted into the substrate, camphor, forming the product, exo-5-hydroxycamphor. Such a branching ratio for label indicates that water is initially released from an unlageled position and illustrates that the general P-450 mixed-function oxidase stoichiometry generates H218O from 18O2 only after multiple-turnover equilibration with the acylating carboxylate oxygen. Formation of an acyl peroxide state is a natural intermediate in peracid, "oxene", or radical mechanisms for methylene carbone oxygenation.
运用同位素示踪法，我们已表明二氢硫辛酸（2,3-硫辛酸）使亚铁氧化态的假单胞菌细胞色素P-450的远端氧发生酰化，形成一种瞬态酰基过氧化物中间体，该中间体有助于氧-氧键的断裂。用18O2进行的单周转研究表明，每有一个氧-18插入底物樟脑形成产物外-5-羟基樟脑时，就有一个氧-18原子掺入硫辛酸的羧基中。这样的标记分支比表明水最初是从未标记的位置释放出来的，并且说明一般的P-450混合功能氧化酶化学计量仅在与酰化羧基氧进行多周转平衡后才从18O2生成H218O。酰基过氧化物状态的形成是过酸、“氧烯”或自由基机制使亚甲基碳氧化过程中的一种天然中间体。
