Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
J Mol Biol. 2010 Jul 16;400(3):518-30. doi: 10.1016/j.jmb.2010.05.035. Epub 2010 May 28.
Yeast flavocytochrome b(2) (Fcb2) is an L-lactate:cytochrome c oxidoreductase in the mitochondrial intermembrane space participating in cellular respiration. Each enzyme subunit consists of a cytochrome b(5)-like heme domain and a flavodehydrogenase (FDH) domain. In the Fcb2 crystal structure, the heme domain is mobile relative to the tetrameric FDH core in one out of two subunits. The monoclonal antibody B2B4, elicited against the holoenzyme, recognizes only the native heme domain in the holoenzyme. When bound, it suppresses the intramolecular electron transfer from flavin to heme b(2), hence cytochrome c reduction. We report here the crystal structure of the heme domain in complex with the Fab at 2.7 A resolution. The Fab epitope on the heme domain includes the two exposed propionate groups of the heme, which are hidden in the interface between the domains in the complete subunit. The structure discloses an unexpected plasticity of Fcb2 in the neighborhood of the heme cavity, in which the heme has rotated. The epitope overlaps with the docking area of the FDH domain onto the heme domain, indicating that the antibody displaces the heme domain in a movement of large amplitude. We suggest that the binding sites on the heme domain of cytochrome c and of the FDH domain also overlap and therefore that cytochrome c binding also requires the heme domain to move away from the FDH domain, so as to allow electron transfer between the two hemes. Based on this hypothesis, we propose a possible model of the Fcb2.cytochrome c complex. Interestingly, this model shares similarity with that of the cytochrome b(5) x cytochrome c complex, in which cytochrome c binds to the surface around the exposed heme edge of cytochrome b(5). The present results therefore support the idea that the heme domain mobility is an inherent component of the Fcb2 functioning.
酵母黄素细胞色素 b(2)（Fcb2）是线粒体内膜间隙中的 L-乳酸：细胞色素 c 氧化还原酶，参与细胞呼吸。每个酶亚基由细胞色素 b(5)-样血红素结构域和黄素脱氢酶（FDH）结构域组成。在 Fcb2 晶体结构中，血红素结构域在两个亚基中的一个相对于四聚体 FDH 核心是可移动的。针对全酶产生的单克隆抗体 B2B4 仅识别全酶中的天然血红素结构域。结合后，它抑制从黄素到血红素 b(2)的分子内电子转移，因此抑制细胞色素 c 的还原。我们在此报告血红素结构域与 Fab 复合物的晶体结构，分辨率为 2.7Å。血红素结构域上的 Fab 表位包括血红素的两个暴露的丙酸盐基团，这些基团在完整亚基的结构域之间的界面中隐藏。该结构揭示了 Fcb2 在血红素腔附近的出乎意料的可塑性，其中血红素已旋转。表位与 FDH 结构域对接血红素结构域的区域重叠，表明抗体以大振幅移动置换血红素结构域。我们假设细胞色素 c 和 FDH 结构域的血红素结构域上的结合位点也重叠，因此细胞色素 c 的结合也需要血红素结构域远离 FDH 结构域，以便在两个血红素之间进行电子转移。基于这一假设，我们提出了 Fcb2.细胞色素 c 复合物的可能模型。有趣的是，该模型与细胞色素 b(5) x 细胞色素 c 复合物的模型相似，其中细胞色素 c 结合到细胞色素 b(5)暴露的血红素边缘周围的表面。因此，这些结果支持血红素结构域的运动是 Fcb2 功能的固有组成部分的观点。
