Reaction of reduced flavins and flavoproteins with diphenyliodonium chloride
Reaction of reduced flavins and flavoproteins with diphenyliodonium chloride
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DOI：
10.1074/jbc.m205432200
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发表时间：
2002-11-01
影响因子：
4.8
通讯作者：
Massey, V
中科院分区：
文献类型：
--
作者：
Chakraborty, S;Massey, V
在厌氧条件下，用停流分光光度法研究了二苯碘氯化铵与游离还原黄素的反应，并确定了二级速率常数与ph的函数关系。根据观察到的反应pK为6.7，确定了活性黄素为还原阴离子。产物混合物不受反应物初始浓度的影响，含有接近20%的氧化黄素。结果可以定量模拟由Tew （Tew， D. G. (1993) Biochemistry 32,10209-10215）提出的机制修改。分析了复合反应混合物的组成，确定了四种具有不同吸光度和荧光特性的黄素-苯基加合物，包括黄素C4a， N5和C8位置的取代。还研究了二苯硫酮对黄蛋白酶的失活作用，并发现了几个例子，尽管在其反应机制中不涉及自由基中间体，但很容易发生失活。由此可以得出结论，苯硫鎓的失活并不是自由基中间体催化机制的有效指标。决定失活的几个因素之一是在稳定的催化状态下维持还原形式的黄素酶，其他因素是氧化还原电位和抑制剂对黄素活性位点的可及性。
The reaction of diphenyliodonium chloride with free reduced flavins has been studied by stopped flow spectrophotometry under anaerobic conditions, and second order rate constants were determined as a function of pH. The reactive flavin species was identified as the reduced anion, based on an observed reaction pK of 6.7. The product mixture was independent of the initial concentration of reactant and contained similar to20% oxidized flavin. The results can be modeled quantitatively on a modification of the mechanism proposed by Tew (Tew, D. G. (1993) Biochemistry 32,10209-10215). The composition of the complex reaction mixture has been analyzed, and four flavin-phenyl adducts with distinctive absorbance and fluorescence characteristics have been identified, involving substitution at the flavin C4a, N5, and C8 positions. Inactivation of flavoprotein enzymes by diphenyliodonium has also been studied, and several examples were found where inactivation occurs readily, despite noninvolvement of radical intermediates in their reaction mechanisms. It can be concluded that inactivation by phenyliodonium species is not a valid indicator of catalytic mechanism involving radical intermediates. One of the several factors determining inactivation is maintenance of the enzyme flavin in the reduced form in the steady state of catalysis, the other factors being redox potential and accessibility of the inhibitor to the flavin active site.