Lehman T C, Thorpe C
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Biochemistry. 1990 Nov 27;29(47):10594-602. doi: 10.1021/bi00499a004.
Medium-chain acyl-CoA dehydrogenase reduced with octanoyl-CoA is reoxidized in two one-electron steps by two molecules of the physiological oxidant, electron transferring flavoprotein (ETF). The organometallic oxidant ferricenium hexafluorophosphate (Fc+PF6-) is an excellent alternative oxidant of the dehydrogenase and mimics a number of the features shown by ETF. Reoxidation of octanoyl-CoA-reduced enzyme (200 microM Fc+PF6- in 100 mM Hepes buffer, pH 7.6, 1 degree C) occurs in two one-electron steps with pseudo-first-order rate constants of 40 s-1 and about 200 s-1 for k1 and k2, respectively. The reaction is comparatively insensitive to ionic strength, and evidence of rate saturation is encountered at high ferricenium ion concentration. As observed with ETF, the free two-electron-reduced dehydrogenase is a much poorer kinetic reductant of Fc+PF6-, with rate constants of 3 s-1 and 0.3 s-1 (for k1 and k2, respectively) using 200 microM Fc+PF6-. In addition to the enoyl-CoA product formed during the dehydrogenation of octanoyl-CoA, binding a number of redox-inert acyl-CoA analogues (notably 3-thia- and 3-oxaoctanoyl-CoA) significantly accelerates electron transfer from the dehydrogenase to Fc+PF6-. Those ligands most effective at accelerating electron transfer favor deprotonation of reduced flavin species in the acyl-CoA dehydrogenase. Thus this rate enhancement may reflect the anticipated kinetic superiority of anionic flavin forms as reductants in outer-sphere electron-transfer processes. Evidence consistent with the presence of two distinct loci for redox communication with the bound flavin in the acyl-CoA dehydrogenase is presented.
与辛酰辅酶A结合而被还原的中链酰基辅酶A脱氢酶，通过两分子生理性氧化剂——电子传递黄素蛋白（ETF），以两个单电子步骤被再氧化。有机金属氧化剂六氟磷酸铁鎓（Fc⁺PF₆⁻）是该脱氢酶的一种优良替代氧化剂，且模拟了ETF所展现的许多特性。辛酰辅酶A还原酶的再氧化（在100 mM Hepes缓冲液，pH 7.6，1℃中加入200 μM Fc⁺PF₆⁻）以两个单电子步骤进行，k₁和k₂的准一级速率常数分别为40 s⁻¹和约200 s⁻¹。该反应对离子强度相对不敏感，并且在高铁鎓离子浓度下会出现速率饱和的迹象。正如用ETF观察到的那样，游离的双电子还原脱氢酶是Fc⁺PF₆⁻的一种更差的动力学还原剂，使用200 μM Fc⁺PF₆⁻时，速率常数分别为3 s⁻¹和0.3 s⁻¹（分别对应k₁和k₂）。除了在辛酰辅酶A脱氢过程中形成的烯酰辅酶A产物外，结合多种氧化还原惰性的酰基辅酶A类似物（特别是3 - 硫杂 - 和3 - 氧杂辛酰辅酶A）会显著加速从脱氢酶到Fc⁺PF₆⁻的电子转移。那些最有效地加速电子转移的配体有利于酰基辅酶A脱氢酶中还原黄素物种的去质子化。因此，这种速率增强可能反映了阴离子黄素形式作为外层电子转移过程中还原剂所预期的动力学优势。本文提供了与酰基辅酶A脱氢酶中与结合黄素进行氧化还原通讯的两个不同位点的存在相一致的证据。
