Mallett T C, Claiborne A
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, USA.
Biochemistry. 1998 Jun 16;37(24):8790-802. doi: 10.1021/bi9803630.
The flavoprotein NADH oxidase (O2 --> 2H2O) from Enterococcus faecalis 10C1 contains a cysteinyl redox center, in addition to FAD. We have proposed a cysteine-sulfenic acid (Cys-SOH) structure for the oxidized form of Cys42; the presence of this redox center is consistent with the stoichiometries reported for earlier reductive titrations of wild-type oxidase, and we have proposed that Cys42-SH plays a key role in the overall four-electron reduction of O2 --> 2H2O. To test these proposals, we provide in this report an analysis of the oxidative half-reaction of an oxidase mutant in which Cys42 is replaced by Ser. NADH titrations lead to direct flavin reduction with 1.05 equiv of NADH/FAD and give rise to the formation of a very stable E-FADH2.NAD+ complex. Kinetic analyses indicate that this species is catalytically competent, and its reactivity with O2 has been analyzed in detail by stopped-flow spectrophotometry using both single-wavelength and diode-array modes of data acquisition. The combined results of this analysis demonstrate that replacement of Cys42 with Ser provides for an altered O2 reduction stoichiometry in which H2O2, not 2H2O, is the product. The two subunits of the reduced enzyme.NAD+ complex react with O2 in an asymmetric mechanism, consistent with an alternating sites cooperativity model such as that proposed [Miller, S. M., Massey, V., Williams, C. H., Jr., Ballou, D. P., and Walsh, C. T. (1991) Biochemistry 30, 2600-2612] for mercuric reductase. An FAD C(4a)-hydroperoxide is identified as the primary oxygenated intermediate in reoxidation of the complex, but the reaction of O2 with the complementary subunit does not proceed until full reoxidation has occurred at the primary subunit. To our knowledge, this is the first report of a C(4a)-peroxyflavin intermediate outside the flavoprotein monooxygenase class.
粪肠球菌10C1的黄素蛋白NADH氧化酶（O₂→2H₂O）除了含有黄素腺嘌呤二核苷酸（FAD）外，还含有一个半胱氨酰氧化还原中心。我们提出了半胱氨酸亚磺酸（Cys-SOH）结构来解释Cys42的氧化形式；这个氧化还原中心的存在与之前报道的野生型氧化酶还原滴定的化学计量一致，并且我们提出Cys42-SH在O₂→2H₂O的整个四电子还原过程中起关键作用。为了验证这些提议，我们在本报告中对一个氧化酶突变体的氧化半反应进行了分析，该突变体中Cys42被Ser取代。NADH滴定导致FAD直接被1.05当量的NADH还原，并形成一个非常稳定的E-FADH₂·NAD⁺复合物。动力学分析表明该物种具有催化活性，并且使用单波长和二极管阵列数据采集模式的停流分光光度法详细分析了其与O₂的反应性。该分析的综合结果表明，用Ser取代Cys42会导致O₂还原化学计量发生改变，产物是过氧化氢（H₂O₂）而非2H₂O。还原酶·NAD⁺复合物的两个亚基以不对称机制与O₂反应，这与交替位点协同模型一致，如之前为汞还原酶所提出的模型[Miller, S. M., Massey, V., Williams, C. H., Jr., Ballou, D. P., and Walsh, C. T. (1991) Biochemistry 30, 2600 - 2612]。在复合物再氧化过程中，FAD C(4a)-氢过氧化物被鉴定为主要的氧化中间体，但O₂与互补亚基的反应直到主要亚基完全再氧化后才进行。据我们所知，这是黄素蛋白单加氧酶类别之外首次报道C(4a)-过氧黄素中间体。
