Pace C, Stankovich M
Biochemistry. 1986 May 6;25(9):2516-22. doi: 10.1021/bi00357a035.
This is the first report of the redox potentials of glycolate oxidase. The pH dependence of the redox behavior as well as the effects of activators and inhibitors was studied. At pH 7.1 in 10 mM imidazole-chloride, Eo1' (EF1ox/EF1-.) is -0.033 +/- 0.010 V and Eo2' (EF1-./EF1redH-) is -0.017 +/- 0.017 V vs. the standard hydrogen electrode at 10 degrees C. A maximum of 29% flavin mononucleotide (FMN) anion radical is stabilized at half-reduction at pH 7.1 and 10 degrees C. Both redox couples of glycolate oxidase are pH-dependent from pH 7 to pH 9, and the FMN anion radical is stabilized in this range. The redox potentials of glycolate oxidase are shifted markedly positive of those of unbound FMN, consistent with the enzyme's function. The midpoint potential of glycolate oxidase is more positive than that of the glyoxalate/glycolate couple, and two-electron reduction of glycolate oxidase is thermodynamically favorable. The redox behavior of glycolate oxidase markedly contrasts that of other flavoprotein oxidases. For most flavoprotein oxidases, Eo1' is independent of pH from pH 7 to pH 9 and is much more positive than Eo2', which is pH-dependent. We present a mechanism that suggests a structural basis for the positive shifts and pH dependence of both Eo1' and Eo2' of glycolate oxidase.
这是关于乙醇酸氧化酶氧化还原电位的首次报道。研究了氧化还原行为的pH依赖性以及激活剂和抑制剂的作用。在10 mM咪唑 - 氯化物缓冲液中，pH 7.1，10℃时，相对于标准氢电极，Eo1'（EF1ox/EF1-.）为 -0.033±0.010 V，Eo2'（EF1-./EF1redH-）为 -0.017±0.017 V。在pH 7.1和10℃半还原时，最多29%的黄素单核苷酸（FMN）阴离子自由基得以稳定。乙醇酸氧化酶的两个氧化还原对在pH 7至pH 9范围内均依赖于pH，且FMN阴离子自由基在此范围内稳定。乙醇酸氧化酶的氧化还原电位明显高于游离FMN的氧化还原电位，这与该酶的功能一致。乙醇酸氧化酶的中点电位比乙醛酸/乙醇酸电对的中点电位更正，乙醇酸氧化酶的双电子还原在热力学上是有利的。乙醇酸氧化酶的氧化还原行为与其他黄素蛋白氧化酶明显不同。对于大多数黄素蛋白氧化酶，Eo1'在pH 7至pH 9范围内与pH无关，且比Eo2'更正，而Eo2'是依赖于pH的。我们提出了一种机制，该机制为乙醇酸氧化酶的Eo1'和Eo2'的正移和pH依赖性提供了结构基础。
