Tsatsos P H, Reynolds K, Nickels E F, He D Y, Yu C A, Gennis R B
School of Chemical Sciences, University of Illinois, Urbana 61801, USA.
Biochemistry. 1998 Jul 14;37(28):9884-8. doi: 10.1021/bi9809270.
The cytochrome bo3 ubiquinol oxidase contains at least one and possibly two binding sites for ubiquinol/ubiquinone. Previous studies used the photoreactive affinity label 3-[3H]azido-2-methyl-5-methoxy-6-geranyl-1,4-benzoquinone (azido-Q), a substrate analogue, to demonstrate that subunit II contributes to at least one of the quinol binding sites. In the current work, mass spectroscopy is used to identify a peptide within subunit II that is photolabeled by the azido-Q. Purified cytochrome bo3 was photolabeled as previously described using azido-Q that was not tritiated (i.e., not radiolabeled). Subunit II was then isolated from an SDS-PAGE gel and proteolyzed in situ with trypsin. The resulting peptides were eluted from the gel and then identified using matrix-assisted laser desorption ionization mass spectrometry. The resulting mass spectrum was compared to that obtained by analysis of subunit II that had not been exposed to the photolabel. Using the amino acid sequence, each peak in the mass spectrum of the unlabeled subunit II could be assigned to an expected trypsin fragment. Two additional peaks were observed in the mass spectrum of the photolabeled subunit with m/z 1931.9 and 2287.7. Subtraction of the mass of azido-Q from the peak at m/z 1931.9 results in a mass equivalent to that of a peptide consisting of amino acids 165-178. The assignment of the peak at m/z 2287.7 cannot be made unequivocally and may correspond either to the covalent attachment of azido-Q to peptide 254-270 or to a peptide resulting from incomplete proteolysis. The labeled peptide, 165-178, is within the water-soluble domain of subunit II, whose X-ray structure is known. This peptide is located near the site where CuA is located in the homologous cytochrome c oxidases and can be placed near the interface between subunits I and II.
细胞色素 bo3 泛醇氧化酶含有至少一个且可能有两个泛醇/泛醌结合位点。先前的研究使用光反应性亲和标记物 3-[3H]叠氮基-2-甲基-5-甲氧基-6-香叶基-1,4-苯醌（叠氮-Q），一种底物类似物，来证明亚基 II 对至少一个泛醇结合位点有贡献。在当前工作中，使用质谱法鉴定亚基 II 中被叠氮-Q 光标记的一种肽段。按照之前描述的方法，用未氚化（即未放射性标记）的叠氮-Q 对纯化的细胞色素 bo3 进行光标记。然后从 SDS-PAGE 凝胶中分离出亚基 II，并在原位用胰蛋白酶进行蛋白水解。将得到的肽段从凝胶中洗脱出来，然后使用基质辅助激光解吸电离质谱法进行鉴定。将得到的质谱图与未暴露于光标记物的亚基 II 分析得到的质谱图进行比较。利用氨基酸序列，未标记的亚基 II 质谱图中的每个峰都可以对应到一个预期的胰蛋白酶片段。在光标记的亚基 II 的质谱图中观察到另外两个峰，质荷比分别为 1931.9 和 2287.7。从质荷比为 1931.9 的峰中减去叠氮-Q 的质量，得到的质量与由氨基酸 165 - １７８ 组成的肽段质量相当。质荷比为 2287.7 的峰的归属不能明确确定，可能对应于叠氮-Q 与肽段 254 - 270 的共价连接，或者对应于不完全蛋白水解产生的一个肽段。被标记的肽段 165 - 178 位于亚基 II 的水溶性结构域内，其 X 射线结构是已知的。该肽段位于同源细胞色素 c 氧化酶中 CuA 所在位点附近，并且可以定位在亚基 I 和亚基 II 之间的界面附近。
