Department of Chemistry, Georgia State University, Atlanta, Georgia 30302-4098, USA.
Biochemistry. 2010 Apr 6;49(13):2952-61. doi: 10.1021/bi902048c.
The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants >or=10(5) M(-1) s(-1) and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 A from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by approximately 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.
黄素还原辅因子被氧氧化是一个非常重要的反应，它是数百种被归类为单加氧酶和氧化酶的黄素蛋白化学多功能性的核心。这些酶的特征是双分子速率常数>或=10(5) M(-1) s(-1)，分别产生水和过氧化氢。先前在单加氧酶的三维结构中已经鉴定出靠近反应性黄素 C(4a)原子的疏水区，但在黄素蛋白氧化酶中没有。在本研究中，我们通过 X 射线晶体学、突变、稳态和快速反应方法研究了 Val464 的作用，Val464 在胆碱氧化酶中距离黄素 C(4a)原子<6 A。Val464Ala 酶的 3D 结构与野生型酶基本相同，如 X 射线晶体学所示。酶促还原的时间分辨厌氧底物表明，用丙氨酸或苏氨酸取代 Val464 不会影响还原半反应。稳态和快速动力学以及酶监测周转表明，Ala464 和 Thr464 酶的氧化半反应相对于野生型酶降低了约 50 倍。我们提出，胆碱氧化酶中的 Val464 侧链提供了一个非极性位点，该位点需要在黄素的 C(4a)原子附近引导氧气，随后通过静电催化进行反应。对现有结构的可视化分析表明，类似的非极性位点可能存在于大多数黄素蛋白氧化酶中。机理考虑为单加氧酶和氧化酶中位点的差异提供了合理化解释。
