Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF.
Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF.
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DOI：
10.1021/bi201508t
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发表时间：
2011-12-20
期刊：
影响因子：
2.9
通讯作者：
Kranz, Robert G.
中科院分区：
文献类型：
--
作者：
Francisco, Brian San;Bretsnyder, Eric C.;Rodgers, Kenton R.;Kranz, Robert G.
在许多细菌、古生菌和植物线粒体中，细胞色素c的成熟涉及到整体膜蛋白CcmF，该蛋白被认为通过促进血红素与细胞色素c的最终共价结合而发挥细胞色素c合成酶的作用。我们之前报道过，大肠杆菌CcmF蛋白含有一种b型血红素，该蛋白稳定且与细胞色素c相关，而不是附着在细胞色素c上的血红素。我们发现，两种保守的跨膜组氨酸（His261或His491）中的任何一种突变都会损害CcmF中b-血红素的化学计量结合，并导致剩余血红素的光谱扰动。外源咪唑能够纠正小侧链（Ala或Gly）取代His261和His491的细胞色素c成熟，这表明在这些CcmF突变体中形成了一个“空腔”，其中咪唑结合并作为b-血红素的功能性配体。野生型CcmF的共振拉曼光谱结果与具有至少一个内源轴向His配体的六配位低自旋b-血红素一致。对多种原核生物纯化的重组CcmF蛋白的分析表明，CcmF中的b-血红素是广泛保守的。我们还测定了CcmF b-血红素的还原电位（Em,7 = -147 mV）。我们在CcmF作为血红素还原酶和细胞色素c合成酶的结构和功能的背景下讨论这些结果。
Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a “cavity” is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (Em,7 = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.
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