Vibrational Characteristics of Mutant and Wild-type Carbon Monoxy Cytochrome c Oxidase: Evidence for a Linear Arrangement of Heme a, a3, and CuB
Vibrational Characteristics of Mutant and Wild-type Carbon Monoxy Cytochrome c Oxidase: Evidence for a Linear Arrangement of Heme a, a3, and CuB
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突变型和野生型单氧碳细胞色素 c 氧化酶的振动特征：血红素 a、a3 和 CuB 线性排列的证据
DOI：
10.1021/ja00091a084
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发表时间：
1994
影响因子：
15
通讯作者：
G. Babcock
中科院分区：
文献类型：
--
作者：
J. Hosler;Younkyoo Kim;J. Shapleigh;R. Gennis;J. O. Alben;S. Ferguson;G. Babcock
将遗传、生化和光谱技术结合应用于需氧生物的关键氧代谢酶细胞色素氧化酶的细菌版本，已经导致了蛋白质框架内血红素和铜辅助因子的拓扑模型。目前的观点认为氧激活中心血红素a3/CuB和低自旋血红素血红素a位于酶的I亚基内。在最新的模型中（图1），两种血红素都结合到跨膜螺旋X上，his02和His421作为血红素a和his419连接血红素a3的配体（见文献1和2）。其余三个保守的组氨酸，His284，-333和334，被指定为CuB配体。位于膜外侧的多肽环连接螺旋IX和X，并包含许多保守残基，包括Asp412。根据模型，Asp412位于血红素a3的近端，His333位于远端，作为CuB的配体。虽然有大量证据支持这些金属配体的指定，但关于His419或His284作为血红素a3配体的指定，存在一些相互矛盾的数据。这个任务决定了立方体是位于两个血红素之间还是远端，因此对理解其功能至关重要。7’8在这里，我们通过分析333和412位置的突变体在未配体完全还原和一氧化碳形式下的振动特性来测试图1的模型。构建球形红杆菌细胞色素aa3突变体，其中I亚基His333或Asp412被天冬酰胺取代（分别为His333Asn和Asp412Asn）
The combined application of genetic, biochemical, andspec-troscopic techniques to bacterial versions of the key oxygenmetabolizing enzyme of aerobic organisms, cytochrome oxidase, has led to topological models of the heme and copper cofactors within the protein framework. 1* 5 The current view places the oxygen-activating center, heme a3/CuB, and the low-spin heme, heme a, within subunit I of the enzyme. In the most recent model (Figure 1), both hemes are bound to transmembrane helix X, with Hisl02 and His421 functioning as the ligands for heme a and His419-ligating heme a3 (see refs 1 and 2). Three remaining conserved histidines, His284,-333, and-334, are assigned as CuB ligands. A polypeptide loop located on the outer side of the membrane connects helices IX and X and contains a number of conserved residues, including Asp412. According to the model, Asp412 is on the proximal side of heme a3, while His333 is on the distal side, as a ligand of CuB. Although there is substantial evidence to support these metal ligand assignments, 1* 5 there have been some conflicting data regarding the designation of His419 or His284 as the ligand of heme a3. 6 This assignment dictates whether CuBis located between or distal to the two hemes, and thus is critical forunderstanding its function. 7'8 Here we have tested the model of Figure 1 by analyzing mutants at the 333 and 412 positions fortheir vibrational properties in both the unliganded fully reduced and carbon monoxy forms. Mutants in cytochrome aa3 of Rhodobacter sphaeroides in which His333 or Asp412 of subunit I was substituted with asparagine (His333Asn and Asp412Asn, respectively) were constructed as