Mass spectrometric analysis of the ubiquinol-binding site in cytochrome bd from Escherichia coli
Mass spectrometric analysis of the ubiquinol-binding site in cytochrome bd from Escherichia coli
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DOI：
10.1074/jbc.m508206200
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发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Mogi, T
中科院分区：
文献类型：
--
作者：
Matsumoto, Y;Murai, M;Mogi, T
细胞色素bd是大肠杆菌需氧呼吸链中的异二聚体末端泛醇氧化酶。为了理解醌醇氧化的独特催化机制，使用质谱法来鉴定可用还原形式的2-叠氮基-3-甲氧基-5-甲基-6-香叶基-1，4-苯醌或2-甲氧基-3-叠氮基-5-甲基-6-香叶基-1，4-苯醌标记的氨基酸残基。基质辅助激光解吸电离飞行时间质谱表明，泛醇-1氧化酶活性的光失活伴随着亚基I与叠氮基喹啉的标记。用赖氨酰内肽酶和内切蛋白酶Asp-N在凝胶内双消化产生的亚基I肽通过反相高效液相色谱法鉴定交联结构域。电喷雾电离四极杆飞行时间质谱法确定肽的氨基酸序列（m/z 1047.5）为Glu（278）-Lys（283），其中叠氮基-Q（2）的光产物连接至I-Glu（280）的羧基侧链。本研究直接证明了周质环VI/VII（Q环）的N-末端区域是醌醇氧化位点的一部分，并表明醌环的2-和3-甲氧基紧邻I-Glu（280）。
Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4- benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4- benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).