Hille R
Department of Medical Biochemistry, Ohio State University, Columbus 43210.
Biochemistry. 1991 Sep 3;30(35):8522-9. doi: 10.1021/bi00099a004.
Solvent kinetic isotope effect studies of electron transfer within xanthine oxidase have been performed, using a stopped-flow pH-jump technique to perturb the distribution of reducing equivalents within partially reduced enzyme and follow the kinetics of reequilibration spectrophotometrically. It is found that the rate constant for electron transfer between the flavin and one of the iron-sulfur centers of the enzyme observed when the pH is jumped from 10 to 6 decreases from 173 to 25 s-1 on going from H2O to D2O, giving an observed solvent kinetic isotope effect of 6.9. An effect of comparable magnitude is observed for the pH jump in the opposite direction, the rate constant decreasing from 395 to 56 s-1. The solvent kinetic isotope effect on kobs is found to be directly proportional to the mole fraction of D2O in the reaction mix for the pH jump in each direction, consistent with the effect arising from a single exchangeable proton. Calculations of the microscopic rate constants for electron transfer between the flavin and the iron-sulfur center indicate that the intrinsic solvent kinetic isotope effect for electron transfer from the neutral flavin semiquinone to the iron-sulfur center designated Fe/S I is substantially greater than for electron transfer in the opposite direction and that the observed solvent kinetic isotope effect is a weighted averaged of the intrinsic isotope effects for the forward and reverse microscopic electron-transfer steps.(ABSTRACT TRUNCATED AT 250 WORDS)
利用停流pH跃变技术来扰动部分还原酶中还原当量的分布，并通过分光光度法跟踪再平衡动力学，开展了黄嘌呤氧化酶内电子转移的溶剂动力学同位素效应研究。研究发现，当pH从10跃变到6时，在从H₂O变为D₂O的过程中，观察到的黄素与酶的一个铁硫中心之间电子转移的速率常数从173 s⁻¹降至25 s⁻¹，观测到的溶剂动力学同位素效应为6.9。在相反方向的pH跃变中也观察到了相当程度的效应，速率常数从395 s⁻¹降至56 s⁻¹。对于每个方向的pH跃变，发现溶剂动力学同位素效应在kobs上与反应混合物中D₂O的摩尔分数成正比，这与单个可交换质子产生的效应一致。黄素与铁硫中心之间电子转移的微观速率常数计算表明，从中性黄素半醌到指定为Fe/S I的铁硫中心的电子转移的固有溶剂动力学同位素效应远大于相反方向的电子转移，并且观测到的溶剂动力学同位素效应是正向和反向微观电子转移步骤固有同位素效应的加权平均值。（摘要截短于250字）
