DEMONSTRATION BY FTIR THAT THE BO-TYPE UBIQUINOL OXIDASE OF ESCHERICHIA-COLI CONTAINS A HEME COPPER BINUCLEAR CENTER SIMILAR TO THAT IN CYTOCHROME-C-OXIDASE AND THAT PROPER ASSEMBLY OF THE BINUCLEAR CENTER REQUIRES THE CYOE GENE-PRODUCT
DEMONSTRATION BY FTIR THAT THE BO-TYPE UBIQUINOL OXIDASE OF ESCHERICHIA-COLI CONTAINS A HEME COPPER BINUCLEAR CENTER SIMILAR TO THAT IN CYTOCHROME-C-OXIDASE AND THAT PROPER ASSEMBLY OF THE BINUCLEAR CENTER REQUIRES THE CYOE GENE-PRODUCT
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DOI：
10.1021/bi00161a023
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发表时间：
1992-11-24
期刊：
影响因子：
2.9
通讯作者：
GENNIS, RB
中科院分区：
文献类型：
--
作者：
HILL, J;GOSWITZ, VC;GENNIS, RB
氨基酸序列数据显示，大肠杆菌的bo型泛醇氧化酶与真核生物的aa 3型细胞色素c氧化酶密切相关。在细胞色素c氧化酶中，氧还原成水发生在由血红素a3和Cu（B）组成的双核中心。本文利用CO与酶结合的傅里叶变换红外光谱（FTIR）直接证明了E。大肠杆菌BO型泛醇氧化酶还含有血红素-铜双核中心。在低温下连接到血红素o的CO的光解（例如，30 K）时形成的CO-Cu络合物，表明存在一个与真核生物氧化酶中血红素a3和Cu（B）形成的双核中心相似的血红素-Cu（B）双核中心。它进一步表明，cyoE基因产物是正确的组装所需的双核中心，虽然这种多肽是不需要作为一个组件的活性酶在体外。cyoE基因产物与COX 10同源，COX 10是来自酿酒酵母的核基因产物，其是酵母细胞色素c氧化酶组装所需的。cyoE基因的缺失导致无活性的醌醇氧化酶，然而，其组装在膜中。结合CO的FTIR分析表明，Cu B存在于该突变体中，但血红素-Cu（B）双核中心是异常的。对膜的血红素含量的分析表明，cyoE缺失导致在双核中心插入血红素B（原血红素IX），而不是血红素O。在该位点插入不正确的血红素似乎是酶无法正常发挥功能的原因。
Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that CUB is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O. The insertion of the incorrect heme at this site appears to be the cause of the inability of the enzyme to function properly.