EXPERIMENTAL AND THEORETICAL-STUDY ON THE REDOX CYCLING OF RESORUFIN BY SOLUBILIZED AND MEMBRANE-BOUND NADPH CYTOCHROME REDUCTASE
EXPERIMENTAL AND THEORETICAL-STUDY ON THE REDOX CYCLING OF RESORUFIN BY SOLUBILIZED AND MEMBRANE-BOUND NADPH CYTOCHROME REDUCTASE
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DOI：
10.1021/tx00026a019
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发表时间：
1992-03-01
影响因子：
4.1
通讯作者：
RIETJENS, IMCM
中科院分区：
文献类型：
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作者：
BALVERS, WG;BOERSMA, MG;RIETJENS, IMCM
本研究描述了nadph -细胞色素还原酶催化间苯二酚氧化还原循环的实验和理论数据。在生理pH值为1-5 μ m的浓度下，乙氧基和己氧基间苯二酚的氧化还原循环比细胞色素P-450依赖的o脱烷基产物间苯二酚（7-羟基苯恶唑）的氧化还原循环效率要高得多。这是由于(i)间苯二酚的质子化形式是一个比去质子化的间苯二酚o阴离子更好的氧化还原循环底物，（ii）在生理pH下，氧化还原循环活性质子化形式仅占间苯二酚总量的1-4%。除实验数据外，AM1分子轨道计算机计算为间苯二酚o阴离子与其质子化形式之间氧化还原循环能力的差异提供了证据。间苯二酚o阴离子的最低未占据分子轨道的能量（E(LUMO)）高于质子化形式的E（LUMO）值。质子化形式的低E（LUMO）值可以作为其更容易还原的参数。此外，计算机计算表明，质子化形式的单电子还原比去质子化的o -阴离子形式的单电子还原在能量上有利363.5 kJ/mol。另外的AM1分子轨道计算机计算表明，单电子还原的间苯二酚会在分子内半醌亚胺部分的o原子上被质子化，然后才有可能被第二个电子还原。最后，通过溶解和膜结合的nadph -细胞色素还原酶对间苯二酚进行氧化还原循环，证明膜环境增加了间苯二酚质子化形式的浓度。这种效果是通过将质子化的间苯二酚分配到膜上和/或通过膜对间苯二酚的质子化平衡的影响来实现的，这有利于质子化的形式。
The present study describes both experimental and theoretical data on the redox cycling of resorufins catalyzed by NADPH-cytochrome reductase. At 1-5-mu-M concentrations at physiological pH, the redox cycling of ethoxy- and pentoxyresorufin was shown to be far more efficient than the redox cycling of their product from the cytochrome P-450 dependent O-dealkylation, resorufin (7-hydroxyphenoxazone). This was shown to result from the fact that (i) the protonated form of the resorufin is a much better substrate for redox cycling than the deprotonated resorufin O-anion and (ii) at physiological pH the redox cycling active protonated form is present at only 1-4% of the total amount of resorufin. In addition to experimental data, AM1 molecular orbital computer calculations provided evidence for the difference in redox cycling capacity between the resorufin O-anion and its protonated form. The energy of the lowest unoccupied molecular orbital (E(LUMO)) of the resorufin O-anion is higher than the E(LUMO) value for the protonated form. This low E(LUMO) value of the protonated form can be taken as a parameter for its easier reduction. Furthermore, computer calculations demonstrated one-electron reduction of the protonated form to be energetically favorable by 363.5 kJ/mol, compared to one-electron reduction of the deprotonated O-anionic form. Additional AM1 molecular orbital computer calculations indicated that the one-electron-reduced resorufin will become protonated at the O-atom of the intramolecular semiquinone imine moiety before reduction by a second electron becomes likely. Finally, redox cycling of resorufin by solubilized and membrane-incorporated NADPH-cytochrome reductase provided evidence that membrane surroundings increase the concentration of the protonated form of resorufin. This effect is achieved either by favored partitioning of the protonated resorufin into the membrane and/or by an effect of the membrane on the protonation equilibrium of resorufin in favor of the protonated form.