Walker M C, Hazzard J T, Tollin G, Edmondson D E
Department of Biochemistry, University of Arizona, Tucson 85721.
Biochemistry. 1991 Jun 18;30(24):5912-7. doi: 10.1021/bi00238a015.
A comparative study using laser flash photolysis of the kinetics of reduction and intramolecular electron transfer among the redox centers of chicken liver xanthine dehydrogenase and of bovine milk xanthine oxidase is described. The photogenerated reductant, 5-deazariboflavin semiquinone, reacts with the dehydrogenase (presumably at the Mo center) in a second-order manner, with a rate constant (k = 6 x 10(7) M-1 s-1) similar to that observed with the oxidase [k = 3 x 10(7) M-1 s-1; Bhattacharyya et al. (1983) Biochemistry 22, 5270-5279]. In the case of the dehydrogenase, neutral FAD radical formation is found to occur by intramolecular electron transfer (kobs = 1600 s-1), presumably from the Mo center, whereas with the oxidase the flavin radical forms via a bimolecular process involving direct reduction by the deazaflavin semiquinone (k = 2 x 10(8) M-1 s-1). Biphasic rates of Fe/S center reduction are observed with both enzymes, which are due to intramolecular electron transfer (kobs approximately 100 s-1 and kobs = 8-11 s-1). Intramolecular oxidation of the FAD radical in each enzyme occurs with a rate constant comparable to that of the rapid phase of Fe/S center reduction. The methylviologen radical, generated by the reaction of the oxidized viologen with 5-deazariboflavin semiquinone, reacts with both the dehydrogenase and the oxidase in a second-order manner (k = 7 x 10(5) M-1 s-1 and 4 x 10(6) M-1 s-1, respectively). Alkylation of the FAD centers results in substantial alterations in the kinetics of the reaction of the viologen radical with the oxidase but not with the dehydrogenase. These results suggest that the viologen radical reacts directly with the FAD center in the oxidase but not in the dehydrogenase, as is the case with the deazaflavin radical. The data support the conclusion that the environments of the FAD centers differ in the two enzymes, which is in accord with other studies addressing this problem from a different perspective [Massey et al. (1989) J. Biol. Chem. 264, 10567-10573]. In contrast, the rate constants for intramolecular electron transfer among the Mo, FAD, and Fe/S centers in the two enzymes (where they can be determined) are quite similar.
本文描述了一项比较研究，该研究利用激光闪光光解技术研究鸡肝黄嘌呤脱氢酶和牛乳黄嘌呤氧化酶氧化还原中心之间的还原动力学及分子内电子转移。光生成的还原剂5-脱氮核黄素半醌以二级反应方式与脱氢酶（可能在钼中心）反应，其速率常数（k = 6×10⁷ M⁻¹ s⁻¹）与氧化酶的速率常数相似[k = 3×10⁷ M⁻¹ s⁻¹；Bhattacharyya等人（1983年），《生物化学》22卷，5270 - 5279页]。对于脱氢酶，发现中性黄素腺嘌呤二核苷酸（FAD）自由基的形成是通过分子内电子转移（观测速率常数kobs = 1600 s⁻¹），推测是从钼中心转移而来，而对于氧化酶，黄素自由基是通过一个双分子过程形成的，该过程涉及脱氮黄素半醌的直接还原（k = 2×10⁸ M⁻¹ s⁻¹）。两种酶都观察到铁硫（Fe/S）中心还原的双相速率，这是由于分子内电子转移（观测速率常数kobs约为100 s⁻¹和kobs = 8 - 11 s⁻¹）。每种酶中FAD自由基的分子内氧化发生的速率常数与Fe/S中心还原的快速相的速率常数相当。氧化态紫精与5-脱氮核黄素半醌反应生成的紫精自由基以二级反应方式与脱氢酶和氧化酶反应（速率常数分别为k = 7×10⁵ M⁻¹ s⁻¹和4×10⁶ M⁻¹ s⁻¹）。FAD中心的烷基化导致紫精自由基与氧化酶反应的动力学发生显著变化，但与脱氢酶反应的动力学没有变化。这些结果表明，紫精自由基直接与氧化酶中的FAD中心反应，而不与脱氢酶中的FAD中心反应，脱氮黄素自由基的情况也是如此。这些数据支持了两种酶中FAD中心环境不同的结论，这与从不同角度研究该问题的其他研究结果一致[Massey等人（1989年），《生物化学杂志》264卷，10567 - 10573页]。相比之下，两种酶中钼、FAD和Fe/S中心之间分子内电子转移的速率常数（在可以测定的情况下）非常相似。
