Harris C M, Massey V
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
J Biol Chem. 1997 Sep 5;272(36):22514-25. doi: 10.1074/jbc.272.36.22514.
Isolated from bovine milk, xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are two interconvertible forms of the same protein, differing in the number of protein cysteines versus cystines. Most differences between XO and XDH are localized to the FAD center, the site at which the oxidizing substrates NAD and molecular oxygen react. A comparative study of the reduction of XO and XDH has been performed to assess differences in reactivity of the molybdopterin site, as well as subsequent electron-transfer events from molybdenum to 2Fe/2S and FAD centers. The compound 4-hydroxypyrimidine (4-OH-P) was chosen as reducing substrate because its higher Km value raised the possibility of binding weak enough to measure kinetically, and its high kcat value could allow detection of intramolecular electron-transfer reactions. As measured by stopped flow spectrophotometry, XO and XDH react with the first equivalent of 4-OH-P via similar mechanisms, differing in the magnitude of rate and dissociation constants. Using [2-2H]4-OH-P as substrate, a D(k/Kd) isotope effect of 1.9 to 2.3 suggests that movement of the hydrogen abstracted from substrate appreciably limits the rate of initial enzyme reduction from Mo(VI) to Mo(IV). Monitoring the visible spectrum of the enzymes, the first observed step is reduction of a single 2Fe/2S center and presumably re-oxidation of Mo(IV) to Mo(V). This suggests a common pathway for electron transfer involving reduction of a 2Fe/2S center prior to reduction of the second 2Fe/2S and FAD centers. Rates of the first electron transfer from molybdenum to the 2Fe/2S center are rapid, 290 s-1 with XO and 180 s-1 with XDH, and are consistent with rates measured by flash photolysis (Walker, M. C., Hazzard, J. T., Tollin, G., and Edmondson, D. E. (1991) Biochemistry 30, 5912-5917) allowing discrete observation of the electron-transfer reactions that occur during turnover. This step also exhibits a modest primary kinetic isotope effect of 1.5 to 1.6 when [2-2H]4-OH-P is used, possibly due to deprotonation of the molybdenum center prior to electron transfer. A second one-electron transfer, presumably oxidizing Mo(V) to Mo(VI), follows in a step coincident with product dissociation, consistent with a role for product release in controlling electron transfer events. The kinetics of this complex system are described and interpreted quantitatively in models that are consistent with all the data.
黄嘌呤氧化酶（XO）和黄嘌呤脱氢酶（XDH）是从牛乳中分离出来的同一蛋白质的两种可相互转化的形式，它们在蛋白质半胱氨酸与胱氨酸的数量上有所不同。XO和XDH之间的大多数差异都集中在FAD中心，即氧化底物NAD和分子氧发生反应的位点。已对XO和XDH的还原进行了比较研究，以评估钼蝶呤位点的反应性差异，以及随后从钼到2Fe/2S和FAD中心的电子转移事件。选择化合物4-羟基嘧啶（4-OH-P）作为还原底物，因为其较高的Km值增加了其结合强度弱到足以进行动力学测量的可能性，并且其较高的kcat值可以检测分子内电子转移反应。通过停流分光光度法测量，XO和XDH通过类似的机制与第一个当量的4-OH-P反应，只是速率和离解常数的大小不同。使用[2-2H]4-OH-P作为底物，1.9至2.3的D(k/Kd)同位素效应表明，从底物中提取的氢的移动明显限制了酶从Mo(VI)还原为Mo(IV)的初始速率。监测酶的可见光谱，首先观察到的步骤是单个2Fe/2S中心的还原，推测是Mo(IV)再氧化为Mo(V)。这表明电子转移存在一条共同途径，即在第二个2Fe/2S和FAD中心还原之前先还原一个2Fe/2S中心。从钼到2Fe/2S中心的第一次电子转移速率很快，XO为290 s-1，XDH为180 s-1，这与通过闪光光解测量的速率一致（Walker, M. C., Hazzard, J. T., Tollin, G., and Edmondson, D. E. (1991) Biochemistry 30, 5912-5917），从而可以离散地观察周转过程中发生的电子转移反应。当使用[2-2H]4-OH-P时，这一步骤还表现出1.5至1.6的适度一级动力学同位素效应，这可能是由于电子转移之前钼中心的去质子化。第二次单电子转移，推测是将Mo(V)氧化为Mo(VI)，紧接着是产物解离步骤，这与产物释放对控制电子转移事件的作用一致。在与所有数据一致的模型中对这个复杂系统的动力学进行了定量描述和解释。
