Verkhovskaya M L, Garcìa-Horsman A, Puustinen A, Rigaud J L, Morgan J E, Verkhovsky M I, Wikström M
Helsinki Bioenergetics Group and Biocentrum Helsinki, Department of Medical Chemistry, Institute of Biomedical Sciences, P.O. Box 8, 00014-University of Helsinki, Helsinki, Finland.
Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10128-31. doi: 10.1073/pnas.94.19.10128.
Glutamic acid 286 (E286; Escherichia coli cytochrome bo3 numbering) in subunit I of the respiratory heme-copper oxidases is highly conserved and has been suggested to be involved in proton translocation. We report a technique of enzyme reconstitution that yields essentially unidirectionally oriented cytochrome bo3 vesicles in which proton translocation can be measured. Such experiments are not feasible in the E286Q mutant due to strong inhibition of respiration, but this is not the case for the mutants E286D and E286C. The reconstituted E286D mutant enzyme readily translocates protons whereas E286C does not. Loss of proton translocation in the D135N mutant, but not in D135E or D407N, also is verified using proteoliposomes. Stopped-flow experiments show that the peroxy intermediate accumulates in the reaction of the E286Q and E286C mutant enzymes with O2. We conclude that an acidic function of the 286 locus is essential for the mechanism of proton translocation.
呼吸血红素 - 铜氧化酶亚基I中的谷氨酸286（E286；大肠杆菌细胞色素bo3编号）高度保守，有人认为它参与质子转运。我们报道了一种酶重组技术，该技术可产生基本上单向取向的细胞色素bo3囊泡，在其中可以测量质子转运。由于对呼吸有强烈抑制作用，在E286Q突变体中进行此类实验不可行，但对于E286D和E286C突变体则并非如此。重组的E286D突变体酶很容易转运质子，而E286C则不能。使用蛋白脂质体也证实了D135N突变体中质子转运的丧失，但D135E或D407N突变体中没有。停流实验表明，过氧中间体在E286Q和E286C突变体酶与O2的反应中积累。我们得出结论，286位点的酸性功能对于质子转运机制至关重要。
