Kaysser T M, Ghaim J B, Georgiou C, Gennis R B
School of Chemical Sciences, University of Illinois, Urbana 61801, USA.
Biochemistry. 1995 Oct 17;34(41):13491-501. doi: 10.1021/bi00041a029.
The cytochrome bd oxidase is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The complex is composed of two subunits (I and II) and three heme prosthetic groups (heme b558, heme b595, and a chlorin, called heme d). Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices. Heme b558 is a six-coordinate, low-spin heme component of the oxidase which has been shown to be contained within subunit I and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane. Previous site-directed mutagenesis studies identified His186, predicted to be near the periplasmic side of transmembrane helix D of subunit I, as one of the axial ligands of heme b558. Since mutagenesis of none of the other histidines in subunit I perturbs heme b558, it was concluded that this heme cannot have bis(histidine) ligation. In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionine residues within subunit I. Among this set of mutants, only the replacement of M393 perturbs heme b558. Replacement of M393 by leucine results in the conversion of heme b558 to a high-spin state. Surprisingly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of the cells. The addition of imidazole to the purified M393L oxidase converts heme b558 back to a low-spin configuration. The data strongly suggest that the sixth axial ligand of heme b558 is methionine-393, and that this heme, therefore, has histidine-methionine ligation. The results are consistent with recent cryogenic near-infrared magnetic circular dichroism spectra that also indicate histidine-methionine ligation of heme b558.
细胞色素bd氧化酶是大肠杆菌有氧呼吸链中的两种末端氧化酶之一。该复合物由两个亚基（I和II）和三个血红素辅基（血红素b558、血红素b595和一种二氢卟酚，称为血红素d）组成。两个亚基都位于细菌细胞质膜内，且每个亚基都有多个假定的跨膜螺旋。血红素b558是氧化酶的一种六配位、低自旋血红素成分，已证明其包含在亚基I中，并与细胞质膜中底物泛醌-8的氧化有关。先前的定点诱变研究确定，预测位于亚基I跨膜螺旋D周质侧附近的His186是血红素b558的轴向配体之一。由于亚基I中其他组氨酸的诱变均未干扰血红素b558，因此得出结论，该血红素不可能有双（组氨酸）配位。在这项工作中，报告了14个突变体的特性，包括亚基I内10个甲硫氨酸残基各自的取代情况。在这组突变体中，只有M393的取代会干扰血红素b558。用亮氨酸取代M393会导致血红素b558转变为高自旋状态。令人惊讶的是，M393L突变并未消除酶活性，且突变体氧化酶有足够的周转能力来支持细胞的有氧生长。向纯化的M393L氧化酶中添加咪唑可使血红素b558恢复为低自旋构型。数据强烈表明，血红素b558的第六个轴向配体是甲硫氨酸-393，因此该血红素有组氨酸-甲硫氨酸配位。这些结果与最近的低温近红外磁圆二色光谱一致，该光谱也表明血红素b558有组氨酸-甲硫氨酸配位。
