Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding.
Laser flash photolysis studies of the kinetics of electron-transfer reactions of Saccharomyces flavocytochrome b2: evidence for conformational gating of intramolecular electron transfer induced by pyruvate binding.
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酵母黄细胞色素 b2 电子转移反应动力学的激光闪光光解研究：丙酮酸结合诱导的分子内电子转移构象门控的证据。
DOI：
10.1021/bi00236a030
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Tollin,G
中科院分区：
文献类型：
--
作者：
Walker,MC;Tollin,G
美国亚利桑那大学生物化学系，图森，亚利桑那州85721摘要：用激光闪光光解法研究了外源去氮黄素半醌类化合物对酿酒酵母中黄细胞色素的还原动力学。去氮黄素半醌对b2血红素和FMN辅因子的直接还原均通过二级动力学进行，速率常数相似（9 X 108 M_1 s_1）。还观察到一个较慢的单指数阶段的FMN再氧化，同时还有一个缓慢的血红素还原阶段。后者约占总血红素吸光度变化的20-25%。这两种慢相都依赖于蛋白质浓度，产生相同的二级速率常数（1.1 X 107 M ' s-1），并且被解释为分子间电子从一个蛋白质分子上的FMN半醌转移到另一个分子上的氧化血红素。与这一结论相一致的是，deflavo1 -flavocytochrome b2没有观察到血红素还原的缓慢阶段。在添加丙酮酸（但不包括d-乳酸或草酸）后，血红素还原的二级速率常数不受影响，但FMN辅助因子的直接还原不再被观察到。血红素辅助因子的还原随后是较慢的部分再氧化，这与FMN还原的单指数阶段同时发生。这两个过程与蛋白质浓度无关，可以解释为分子内电子从还原的b2血红素转移到氧化的FMN的结果。在丙酮酸不存在和不存在的情况下，黄细胞色素的电位滴定表明，在丙酮酸不存在的情况下，电子从FMN转移到血红素的热力学驱动力要大得多。尽管如此，分子内的电子转移只在丙酮酸存在的情况下才被观察到。这一结果是根据丙酮酸结合引起的构象变化来解释的，它允许电子在辅因子之间转移。在丙酮酸存在的情况下，分子内电子转移的速率常数取决于离子强度，表明静电效应的发生影响了这一过程。^东黄细胞色素b2 （l -乳酸脱氢酶，EC1.1）2.3)是一种双功能酶（Jacq & Lederer, 1974），它催化l -乳酸的双电子氧化生成丙酮酸，随后单电子转移到两个细胞色素c
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Received August 23, 1990; Revised Manuscript Received March 5, 1991 abstract: The kinetics of reduction of the flavocytochrome from Saccharomyces cerevisiae by exogenous deazaflavin semiquinones have been investigated by using laser flash photolysis. Direct reduction by deazaflavin semiquinone of both the b2 heme and the FMN cofactor occurred via second-order kinetics with similar rateconstants (9 X 108 M_1 s_1). A slower, monoexponential, phase of FMN reoxidation was also observed, concurrent with a slow phase of heme reduction. The latter accounted for approximately 20-25% of the total heme absorbance change. Both of these slow phases were protein concentration dependent, yielding indentical second-order rate constants (1.1 X 107 M" 1 s-1), and were interpreted as resulting from intermolecular electron transfer from the FMN semiquinone on one protein molecule to an oxidized heme on a second molecule. Consistent with this conclusion, no slow phase of heme reduction was observed with deflavo-flavocytochrome b2. Upon the addition of pyruvate (but not D-lactate or oxalate), the second-order rate constant for heme reduction was unaffected, but direct reduction of the FMN cofactor was no longer observed. Reduction of the heme cofactor was followed by a slower partial reoxidation, which occurred concomitantly with a monoexponential phase of FMN reduction. Both processes were protein concentration independent and were interpreted as the result of intramolecular electron transfer from reduced b2 heme to oxidized FMN. Potentiometric titrations of the flavocytochrome in the absence and presence of pyruvate demonstrated that the thermodynamic driving force for electron transfer from FMN to heme is much greater in the absence of pyruvate. Despite this, intramolecular electron transfer was only observed in the presence of pyruvate. This result is interpreted in terms of a conformational change induced by pyruvatebinding which permits electron transfer between the cofactors. The rate constant for intramolecular electron transfer in the presence of pyruvate was dependent on ionic strength, suggesting the occurrence of electrostatic effects which influence this process.^ east flavocytochrome b2 (L-lactate dehydrogenase, EC1.1. 2.3) is a bifunctional enzyme (Jacq & Lederer, 1974) that catalyzes the two-electron oxidation of L-lactate to pyruvate, and subsequent one-electron transfers to two cytochrome c