Backgren C, Hummer G, Wikström M, Puustinen A
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, P.O. Box 8, 00014 Helsinki, Finland.
Biochemistry. 2000 Jul 11;39(27):7863-7. doi: 10.1021/bi000806b.
A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.
血红素-铜氧化酶亚基I中的一个谷氨酸残基高度保守，并且直接参与了这些呼吸酶的氧气还原和质子泵浦机制。将其突变为天冬氨酸以外的残基会显著抑制活性，质子转运也会丧失。然而，在一些结构上差异较大的血红素-铜氧化酶成员中，这个谷氨酸被一个非酸性残基取代，这些成员在附近有一个酪氨酸残基。在这里，我们利用反硝化副球菌的细胞色素c氧化酶表明，将谷氨酸及其附近一个保守的甘氨酸替换后，催化活性降低到野生型值的<0.1%。但是，如果此外，结构上靠近的一个苯丙氨酸被改变为酪氨酸，活性会提高100倍以上，并且质子转运得以恢复。分子动力学模拟表明，酪氨酸可以支持一个可能对血红素-铜氧化酶中的质子转移至关重要的瞬态水分子阵列。令人惊讶的是，因此谷氨酸并非不可或缺，这对这些酶的催化机制施加了重要限制。
