Mowat C G, Miles C S, Munro A W, Cheesman M R, Quaroni L G, Reid G A, Chapman S K
Department of Chemistry, University of Edinburgh, UK.
J Biol Inorg Chem. 2000 Oct;5(5):584-92. doi: 10.1007/s007750000141.
Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity. As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate. Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation. In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like. Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.
将黄素细胞色素b2（酿酒酵母中的L-乳酸：细胞色素c氧化还原酶）的一个血红素铁轴向配体（His66）用半胱氨酸替代，产生了一种酶（H66C-b2），它仍然是一种活性L-乳酸脱氢酶（kcat为272±6 s(-1)，L-乳酸的KM为0.60±0.06 mM，25℃，离子强度0.10，Tris-HCl，pH 7.5），但没有细胞色素c还原酶活性。由于该突变，发现血红素的还原电位为-265±5 mV，比野生型酶的还原电位负超过240 mV，因此不能被L-乳酸还原。表面增强共振拉曼光谱表明H66C-b2的血红素与细胞色素P450的血红素有相似之处，在1345 cm(-1)处有一个ν4带，这表明是半胱氨酸与血红素铁的配位。此外，电子顺磁共振光谱产生的g值分别为2.33、2.22和1.94，这是低自旋铁细胞色素P450的典型值，光谱显示在600至900 nm之间有特征峰，这是血红素铁硫配位的特征，磁圆二色光谱显示相对于野生型有一个蓝移的近红外电荷转移带，这意味着H66C-b2的血红素类似细胞色素P450。有趣的是，电子顺磁共振证据还表明突变酶中第二个组氨酸血红素铁配体（His43）被取代。
