Wang R, Thorpe C
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Biochemistry. 1991 Aug 13;30(32):7895-901. doi: 10.1021/bi00246a004.
The free two-electron-reduced form of medium-chain acyl-CoA dehydrogenase is reoxidized by 120 microM molecular oxygen (50 mM phosphate buffer, pH 7.6, 2 degrees C) with a half-time of approximately 7 s. Reoxidation yields hydrogen peroxide as a major product with only traces of the superoxide anion. In contrast, enzyme reduced with octanoyl-CoA is extremely slowly reoxidized oxygen, and so a series of 14 different substrate analogues have been tested to assess the structural factors responsible for this effect. Complexes with redox-inactive ligands such as 3-thia- and 2-azaoctanoyl-CoA lead to an approximately 3000-fold slowing of the rate of reoxidation of the free dihydroflavin form of the enzyme. Comparable ligands lacking the thioester carbonyl function are much less effective with rates some 1.3-4-fold slower than the free enzyme. The strong suppression of oxygen reactivity observed with certain ligands is probably not simply a steric effect but may reflect desolvation of the active site and consequent destabilization of the superoxide anion intermediate formed during reoxidation of the flavin. The profound differences in oxygen reactivity between acyl-CoA dehydrogenase and acyl-CoA oxidase and the unusual stability of certain flavoprotein semiquinones in air are discussed in terms of these thermodynamic and kinetic arguments.
中链酰基辅酶A脱氢酶的游离双电子还原形式在120微摩尔分子氧（50毫摩尔磷酸盐缓冲液，pH 7.6，2℃）存在下被再氧化，半衰期约为7秒。再氧化产生过氧化氢作为主要产物，仅产生微量超氧阴离子。相比之下，用辛酰辅酶A还原的酶被氧再氧化的速度极慢，因此已测试了14种不同的底物类似物，以评估造成这种效应的结构因素。与氧化还原无活性配体（如3-硫杂和2-氮杂辛酰辅酶A）形成的复合物会使酶的游离二氢黄素形式的再氧化速率减慢约3000倍。缺乏硫酯羰基功能的类似配体效果要差得多，其速率比游离酶慢约1.3至4倍。某些配体对氧反应性的强烈抑制可能不仅仅是空间效应，还可能反映了活性位点的去溶剂化以及黄素再氧化过程中形成的超氧阴离子中间体的稳定性降低。根据这些热力学和动力学观点，讨论了酰基辅酶A脱氢酶和酰基辅酶A氧化酶在氧反应性上的深刻差异以及某些黄素蛋白半醌在空气中的异常稳定性。
