Maklashina E, Cecchini G
Molecular Biology Division (151-S), VA Medical Center, San Francisco, California, 94121, USA.
Arch Biochem Biophys. 1999 Sep 15;369(2):223-32. doi: 10.1006/abbi.1999.1359.
Escherichia coli succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate reductase (QFR) are excellent model systems to understand the function of eukaryotic Complex II. They have structural and catalytic properties similar to their eukaryotic counterpart. An exception is that potent inhibitors of mammalian Complex II, such as thenoyltrifluoroacetone and carboxanilides, only weakly inhibit their bacterial counterparts. This lack of good inhibitors of quinone reactions and the higher level of side reactions in the prokaryotic enzymes has hampered the elucidation of the mechanism of quinone oxidation/reduction in E. coli Complex II. In this communication DT-diaphorase and an appropriate quinone are used to measure quinol-fumarate reductase activity and E. coli bo-oxidase and quinones are used to determine succinate-quinone reductase activity. Simple Michaelis kinetics are observed for both enzymes with ubiquinones and menaquinones in the succinate oxidase (forward) and fumarate reductase (reverse) reactions. The comparison of E. coli SQR and QFR demonstrates that 2-n-heptyl 4-hydroxyquinoline-N-oxide (HQNO) is a potent inhibitor of QFR in both assays; however, SQR is not sensitive to HQNO. A series of 2-alkyl-4,6-dinitrophenols and pentachlorophenol were found to be potent competitive inhibitors of both SQR and QFR. In addition, the isolated E. coli SQR complex demonstrates a mixed-type inhibition with carboxanilides, whereas the QFR complex is resistant to this inhibitor. The kinetic properties of SQR and QFR suggest that either ubiquinone or menaquinone operates at a single exchangeable site working in forward or reverse reactions. The pH activity profiles for E. coli QFR and SQR are similar showing maximal activity between pH 7.4 and 7.8, suggesting the importance of similar catalytic groups in quinol deprotonation and oxidation.
大肠杆菌琥珀酸 - 泛醌氧化还原酶（SQR）和甲萘醌 - 富马酸还原酶（QFR）是理解真核生物复合物II功能的优秀模型系统。它们具有与其真核对应物相似的结构和催化特性。一个例外是，哺乳动物复合物II的有效抑制剂，如噻吩甲酰三氟丙酮和羧酰苯胺，对其细菌对应物的抑制作用较弱。缺乏有效的醌反应抑制剂以及原核酶中较高水平的副反应阻碍了对大肠杆菌复合物II中醌氧化/还原机制的阐明。在本通讯中，DT - 黄递酶和合适的醌用于测量喹醇 - 富马酸还原酶活性，大肠杆菌bo - 氧化酶和醌用于测定琥珀酸 - 醌还原酶活性。在琥珀酸氧化酶（正向）和富马酸还原酶（反向）反应中，两种酶与泛醌和甲萘醌反应均呈现简单的米氏动力学。大肠杆菌SQR和QFR的比较表明，在两种测定中，2 - 正庚基 - 4 - 羟基喹啉 - N - 氧化物（HQNO）是QFR的有效抑制剂；然而，SQR对HQNO不敏感。发现一系列2 - 烷基 - 4,6 - 二硝基苯酚和五氯苯酚是SQR和QFR的有效竞争性抑制剂。此外，分离的大肠杆菌SQR复合物对羧酰苯胺表现出混合型抑制，而QFR复合物对该抑制剂具有抗性。SQR和QFR的动力学特性表明，泛醌或甲萘醌在正向或反向反应中在单个可交换位点起作用。大肠杆菌QFR和SQR的pH活性曲线相似，在pH 7.4至7.8之间显示出最大活性，表明在喹醇去质子化和氧化中类似催化基团的重要性。
