Mallett T C, Parsonage D, Claiborne A
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
Biochemistry. 1999 Mar 9;38(10):3000-11. doi: 10.1021/bi9817717.
Recent studies [Mallett, T. C., and Claiborne, A. (1998) Biochemistry 37, 8790-8802] of the O2 reactivity of C42S NADH oxidase (O2 --> H2O2) revealed an asymmetric mechanism in which the two FADH2.NAD+ per reduced dimer display kinetic inequivalence. In this report we provide evidence indicating that the fully active, recombinant wild-type oxidase (O2 --> 2H2O) displays thermodynamic inequivalence between the two active sites per dimer. Using NADPH to generate the free reduced wild-type enzyme (EH2'/EH4), we have shown that NAD+ titrations lead to differential behavior as only one FADH2 per dimer binds NAD+ tightly to give the charge-transfer complex. The second FADH2, in contrast, transfers its electrons to the single Cys42-sulfenic acid (Cys42-SOH) redox center, which remains oxidized during the reductive titration. Titrations of the reduced NADH oxidase with oxidized 3-acetylpyridine and 3-aminopyridine adenine dinucleotides further support the conclusion that the two FADH2 per dimer in wild-type enzyme can be described as distinct "charge-transfer" and "electron-transfer" sites, with the latter site giving rise to either intramolecular (Cys42-SOH) or bimolecular (pyridine nucleotide) reduction. The reduced C42S mutant is not capable of intramolecular electron transfer on binding pyridine nucleotides, thus confirming that the Cys42-SOH center is in fact the source of the redox asymmetry observed with wild-type oxidase. These observations on the role of Cys42-SOH in the expression of thermodynamic inequivalence as observed in wild-type NADH oxidase complement the previously described kinetic inequivalence of the C42S mutant; taken together, these results provide the overlapping framework for an alternating sites cooperativity model of oxidase action.
近期关于C42S NADH氧化酶（O2→H2O2）与O2反应性的研究[马利特，T.C.，和克莱伯恩，A.（1998年）《生物化学》37卷，8790 - 8802页]揭示了一种不对称机制，即每个还原二聚体中的两个FADH2·NAD+表现出动力学不等效性。在本报告中，我们提供的证据表明，完全活性的重组野生型氧化酶（O2→2H2O）在每个二聚体的两个活性位点之间表现出热力学不等效性。利用NADPH生成游离的还原野生型酶（EH2'/EH4），我们已经表明，NAD+滴定导致不同的行为，因为每个二聚体中只有一个FADH2紧密结合NAD+形成电荷转移复合物。相比之下，第二个FADH2将其电子转移到单个半胱氨酸42 - 亚磺酸（Cys42 - SOH）氧化还原中心，该中心在还原滴定过程中保持氧化状态。用氧化的3 - 乙酰吡啶和3 - 氨基吡啶腺嘌呤二核苷酸对还原的NADH氧化酶进行滴定，进一步支持了这样的结论：野生型酶中每个二聚体的两个FADH2可被描述为不同的“电荷转移”和“电子转移”位点，后者位点导致分子内（Cys42 - SOH）或双分子（吡啶核苷酸）还原。还原的C42S突变体在结合吡啶核苷酸时不能进行分子内电子转移，从而证实Cys42 - SOH中心实际上是野生型氧化酶中观察到的氧化还原不对称性的来源。这些关于Cys42 - SOH在野生型NADH氧化酶中热力学不等效性表达中作用的观察结果，补充了先前描述的C42S突变体的动力学不等效性；综合起来，这些结果为氧化酶作用的交替位点协同模型提供了重叠框架。
