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.

译文

用辛酰辅酶a还原的中链酰基辅酶a脱氢酶在两个单电子步骤中被两分子生理氧化剂电子转移黄素蛋白 (ETF) 重新氧化。有机金属氧化剂六氟磷酸铁 (Fc PF6-) 是脱氢酶的出色替代氧化剂,并模仿了ETF显示的许多特征。辛酰基辅酶a还原酶 (200 microM Fc + PF6-在100 mM Hepes缓冲液中,pH 7.6,1 ℃) 的再氧化在两个单电子步骤中发生,k1和k2的伪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,在高铁离子浓度下会遇到速率饱和的证据。如用ETF观察到的,游离的双电子还原脱氢酶是Fc + PF6-的差得多的动力学还原剂,使用200 microM Fc + PF6-的速率常数为3 s-1和0.3 s-1 (分别为k1和k2)。除了在辛酰基-CoA脱氢过程中形成的烯酰辅酶a产物外,结合许多氧化还原惰性酰基-CoA类似物 (尤其是3-thia-和3-氧辛酰基-CoA) 还显着加速了电子转移从脱氢酶到Fc PF6-。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,这种速率提高可能反映了阴离子黄素形式作为外球电子转移过程中还原剂的预期动力学优势。提出了与酰基辅酶a脱氢酶中与结合的黄素进行氧化还原通讯的两个不同基因座相一致的证据。

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