Membrane Protein Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
PLoS One. 2012;7(2):e32641. doi: 10.1371/journal.pone.0032641. Epub 2012 Feb 29.
The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.
大肠杆菌琥珀酸脱氢酶中血红素 b 的作用非常模糊，其在催化中的作用也存在疑问。为了研究血红素还原是否是催化机制的必要步骤，我们在血红素结合口袋周围生成了一系列定点突变，创建了一个突变文库，其中血红素的中点电位从野生型的 +20 mV 逐渐降低到 -80 mV，逐步降低。这种中点电位的差异足以改变血红素与琥珀酸的反应性，从而改变其在周转条件下的氧化还原状态。我们的结果表明，酶的稳态琥珀酸氧化酶和延胡索酸还原酶的催化活性都不是血红素氧化还原电位的函数。此外，较低的血红素电位不会在体外和体内引起超氧化物产生速率的增加。野生型酶中血红素的电子顺磁共振（EPR）谱是两个不同信号的组合。我们将 EPR 谱与结构联系起来，表明其中一个信号可能来自于血红素的面外扭曲，即马鞍形构象，而第二个信号则源自卟啉环的更平面取向。
