Pace C P, Stankovich M T
Biochim Biophys Acta. 1987 Feb 25;911(3):267-76. doi: 10.1016/0167-4838(87)90067-7.
Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii catalyzes electron transfer from NADH or D-lactate dehydrogenase to butyryl-CoA dehydrogenase. As a basis for understanding the interactions of ETF with its substrates, we report here on the redox properties of ETF alone. ETF exhibited reversible, two-electron transfer during electrochemical reduction in the presence of mediator dyes. The midpoint redox potentials of the FAD cofactor were -0.185 V at pH 5.5, -0.259 V at pH 7.1 and -0.269 +/- 0.013 V at pH 8.4, all versus the standard hydrogen electrode In the presence of the indicator dye 1-deazariboflavin, the Nernst slopes were 0.029 V and 0.026 V at pH 5.5 and pH 7.1, respectively, compared with an expected value of 0.028 V at 10 degrees C. At pH 8.4, in the presence of 2-hydroxy-1,4-naphthoquinone or phenosafranine, the Nernst slope varied from 0.021 V to 0.041 V. In the experiments at pH 8.4, equilibration was very slow in the reductive direction and a difference of as much as 30 mV was observed between reductive and oxidative midpoints. ETF exhibited no thermodynamic stabilization of the radical form of the FAD cofactor during electrochemical reduction at pH 5.5, 7.1 or 8.4. However, up to 93% of kinetically stable, anionic radical was produced by dithionite titration at pH 8.5. Molar absorptivities of ETF radical were 17,000 M-1 X cm-1 at 365 nm and 5100 M-1 X cm-1 at 450 nm. The four ETF preparations used here contained less than 7% 6-OH-FAD. However, two of the preparations contained significant amounts (up to 30%) of flavin which stabilized radical and reduced at potentials 0.2 V more positive than those required for reduction of the major form of ETF. This is referred to as the B form of ETF. The proportion of ETF-FAD in the B form was increased by incubation with free FAD or by a cycle of reduction and reoxidation. These treatments caused marked changes in the absorption spectrum of oxidized ETF and decreases of 20-25% in ETF units/A450.
来自厌氧细菌埃氏巨球形菌的电子传递黄素蛋白（ETF）催化电子从NADH或D-乳酸脱氢酶转移至丁酰辅酶A脱氢酶。作为理解ETF与其底物相互作用的基础，我们在此报告单独的ETF的氧化还原特性。在存在介体染料的情况下进行电化学还原时，ETF表现出可逆的双电子转移。FAD辅因子的中点氧化还原电位在pH 5.5时为-0.185 V，在pH 7.1时为-0.259 V，在pH 8.4时为-0.269±0.013 V，均相对于标准氢电极。在存在指示剂染料1-去氮杂核黄素的情况下，在pH 5.5和pH 7.1时能斯特斜率分别为0.029 V和0.026 V，而在10℃时预期值为0.028 V。在pH 8.4时，在存在2-羟基-1,4-萘醌或番红精的情况下，能斯特斜率在0.021 V至0.041 V之间变化。在pH 8.4的实验中，还原方向的平衡非常缓慢，并且在还原和氧化中点之间观察到高达30 mV的差异。在pH 5.5、7.1或8.4进行电化学还原期间，ETF对FAD辅因子的自由基形式没有热力学稳定作用。然而，在pH 8.5通过连二亚硫酸盐滴定产生了高达93%的动力学稳定的阴离子自由基。ETF自由基在365 nm处的摩尔吸光系数为17,000 M-1·cm-1，在450 nm处为5100 M-1·cm-1。这里使用的四种ETF制剂中6-OH-FAD含量低于7%。然而，其中两种制剂含有大量（高达30%）的黄素，其能稳定自由基并在比还原主要形式的ETF所需电位更正0.2 V的电位下还原。这被称为ETF的B形式。通过与游离FAD孵育或通过还原和再氧化循环，ETF-FAD中B形式的比例增加。这些处理导致氧化态ETF的吸收光谱发生明显变化，并且ETF单位/A450降低20 - 25%。
