Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.
Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.
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DOI：
10.1021/bi500704p
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发表时间：
2014-08-19
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：
Babbitt, Shalon E.;San Francisco, Brian;Bretsnyder, Eric C.;Kranz, Robert G.
C型细胞色素的特征在于血红素辅因子的共价连接，这是其随后的折叠、稳定性和功能通常所需的修饰。血红素附着发生在线粒体膜间隙，在大多数真核生物中，是由全细胞色素c合成酶（HCCS）介导的。HCCS是真核细胞色素c生物合成途径（称为系统III）的主要组成部分。HCCS的催化功能取决于其协调其底物血红素和细胞色素c之间相互作用的能力。本研究表明，HCCS重组表达和纯化的最新进展促进了对HCCS中保守残基作用的全面分析。在此之前，我们提出了一个四步模型描述HCC介导的细胞色素c组装，确定一个保守的组氨酸残基（His 154）作为轴向配体的血红素铁。在这项研究中，我们进行了系统的突变分析的17个保守残基HCCS，我们提供的证据表明，该酶含有两个血红素结合域。我们的数据表明，血红素接触介导的残基在这些领域内调节血红素结合的动力学，并有助于稳定的HCCS-血红素-细胞色素c稳态三元复合物。虽然一些残基对于初始血红素结合（步骤1）是必需的，但其他残基影响随后的全细胞色素c产物的释放（步骤4）。某些HCCS突变体，血红素结合缺陷的纠正功能的外源性氨基乙酰丙酸（ALA，血红素的前体）。这种化学“校正”支持血红素结合相应残基的拟议作用。
C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS–heme–cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical “correction” supports the proposed role of heme binding for the corresponding residues.
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影响因子：
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通讯作者：
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