Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Biochemistry. 2009 Mar 3;48(8):1820-8. doi: 10.1021/bi801609a.
Maturation of c-type cytochromes in many bacterial species and plant mitochondria requires the participation of the heme chaperone CcmE that binds heme covalently via a His residue (H130 in Escherichia coli) before transferring it stereospecifically to the apo form of cytochromes c. Only the structure of the apo form of CcmE is known; the heme-binding site has been modeled on the surface of the protein in the vicinity of H130. We have determined the reduction potential of CcmE, which suggests that heme bound to CcmE is not as exposed to solvent as was initially thought. Alanine insertions in the vicinity of the heme-binding histidine (which we showed by NMR do not perturb the protein fold) strikingly abolish formation of both holo-CcmE and cytochrome c, whereas previously reported point mutations of residues adjacent to H130 gave only a partial attenuation. The heme iron coordinating residue Y134 proved to be strictly required for axial ligation of both ferrous and ferric heme. These results indicate the existence of a conformationally well-defined heme pocket that involves amino acids located in the proximity of H130. However, mutation of Y134 affected neither heme attachment to CcmE nor cytochrome c maturation, suggesting that heme binding and release from CcmE are hydrophobically driven and relatively indifferent to axial ligation.
许多细菌物种和植物线粒体中的 c 型细胞色素的成熟需要血红素伴侣蛋白 CcmE 的参与，该蛋白通过 His 残基（大肠杆菌中的 H130）共价结合血红素，然后将其立体特异性地转移到细胞色素 c 的脱辅基形式。目前仅已知 CcmE 的脱辅基形式的结构；血红素结合位点已在 H130 附近的蛋白质表面进行建模。我们已经确定了 CcmE 的还原电位，这表明与 CcmE 结合的血红素不像最初认为的那样暴露于溶剂中。血红素结合组氨酸附近的丙氨酸插入（通过 NMR 我们证明这不会干扰蛋白质折叠）显著地阻止了全血红素 CcmE 和细胞色素 c 的形成，而先前报道的 H130 附近残基的点突变仅导致部分衰减。血红素铁配位残基 Y134 被证明严格需要亚铁和高铁血红素的轴向配位。这些结果表明存在一个构象上定义良好的血红素口袋，涉及到位于 H130 附近的氨基酸。然而，Y134 的突变既不影响 CcmE 与血红素的结合，也不影响细胞色素 c 的成熟，这表明血红素结合和从 CcmE 释放是由疏水性驱动的，并且与轴向配位相对无关。
