Paudel H K, Yu L, Yu C A
Department of Biochemistry, Oklahoma Agriculture Experimental Station, Oklahoma State University, Stillwater 74078.
Biochim Biophys Acta. 1991 Jan 22;1056(2):159-65. doi: 10.1016/s0005-2728(05)80282-8.
The involvement of a histidine residue of the membrane-anchoring protein (QPs) fraction in reconstitution of succinate dehydrogenase to form succinate-ubiquinone reductase is studied by using a histidine-modifying reagent, diethylpyrocarbonate (DEPC). A maximum inactivation of 80% of reconstitutive activity is obtained when QPs is treated with 1 mM DEPC at 0 degrees C for 30 min in 50 mM Tris-HCl (pH 7.0). DEPC also inactivates about 85% of intact succinate-ubiquinone reductase. The inactivation of succinate-ubiquinone reductase by DEPC is a result of the modification of essential histidine residues of succinate dehydrogenase. The inactivation is not a result of the modification of the histidine residue in QPs which is essential for interaction with succinate dehydrogenase because the QPs dissociated from the inactivated succinate-ubiquinone reductase is active in reconstitution with active succinate-dehydrogenase. Apparently, the essential histidine in QPs is shielded by succinate dehydrogenase and thus inaccessible to DEPC modification in succinate-ubiquinone reductase. The involvement of a histidine residue of QPs in interaction with succinate dehydrogenase is further evident by the presence of 553 nm shoulder on the alpha-absorption peak of reduced cytochrome b-560 (a characteristic of physical association of QPs with succinate dehydrogenase) in the DEPC-inactivated succinate-ubiquinone reductase. This shoulder disappears from a mixture of succinate dehydrogenase and DEPC-treated QPs when reduced with dithionite. About one histidine residue per molecule of QPs is modified in the DEPC-treated sample, suggesting that only one histidine residue is essential for interaction with succinate dehydrogenase. This essential histidine group is located in the smaller subunit (Mr 13,000) of QPs.
通过使用组氨酸修饰试剂焦碳酸二乙酯（DEPC），研究了膜锚定蛋白（QPs）组分中的组氨酸残基在琥珀酸脱氢酶重构形成琥珀酸-泛醌还原酶过程中的作用。当在50 mM Tris-HCl（pH 7.0）中，于0℃用1 mM DEPC处理QPs 30分钟时，重构活性的最大失活率达到80%。DEPC还能使完整的琥珀酸-泛醌还原酶失活约85%。DEPC使琥珀酸-泛醌还原酶失活是琥珀酸脱氢酶必需组氨酸残基被修饰的结果。失活并非由于QPs中对与琥珀酸脱氢酶相互作用至关重要的组氨酸残基被修饰，因为从失活的琥珀酸-泛醌还原酶上解离的QPs在与活性琥珀酸脱氢酶重构时具有活性。显然，QPs中的必需组氨酸被琥珀酸脱氢酶屏蔽，因此在琥珀酸-泛醌还原酶中无法被DEPC修饰。在DEPC失活的琥珀酸-泛醌还原酶中，还原型细胞色素b-560的α吸收峰上出现553 nm肩峰（这是QPs与琥珀酸脱氢酶物理缔合的特征），进一步证明了QPs的组氨酸残基参与了与琥珀酸脱氢酶的相互作用。当用连二亚硫酸盐还原时，该肩峰在琥珀酸脱氢酶和DEPC处理的QPs混合物中消失。在DEPC处理的样品中，每分子QPs约有一个组氨酸残基被修饰，这表明只有一个组氨酸残基对于与琥珀酸脱氢酶的相互作用是必需的。这个必需的组氨酸基团位于QPs的较小亚基（Mr 13,000）中。
