Electron and proton transfer in the arginine-54-methionine mutant of cytochrome c oxidase from Paracoccus denitrificans
Electron and proton transfer in the arginine-54-methionine mutant of cytochrome c oxidase from Paracoccus denitrificans
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DOI：
10.1021/bi002948b
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发表时间：
2001-05-01
期刊：
影响因子：
2.9
通讯作者：
Wikström, M
中科院分区：
文献类型：
--
作者：
Jasaitis, A;Backgren, C;Wikström, M
反硝化副球菌细胞色素c氧化酶I亚基中的精氨酸54与血红素a的甲酰基相互作用。该精氨酸突变为甲硫氨酸（R54M）可显著改变血红素a的光谱特性并降低其中点氧化还原电位[kant et al .,(1999)]。化学学报，27 (2),391 - 391；Lee et al, (2000) Biochemistry 39, 2989-2996；Riistama et al.（2000）生物化学。Biophys。学报，1456,1-4]。在突变酶的厌氧还原过程中，一小部分血红素a首先与血红素a一起被还原(3)，而大部分血红素a在随后被还原。这表明，由于血红素a的低氧化还原电位，电子转移在热力学上受到了损害，但与野生型酶一样，电子转移仍然从CUA通过血红素a到达双核位点，没有检测到Cu-A直接到双核位点的旁路。与此一致的是，突变酶的质子易位效率在1 H+/e（-）时不受影响，尽管翻转被强烈抑制。时间分辨电滴定法表明，当完全还原酶与O-2反应时，PR -> F转变过程中膜电位生成的快速阶段不受突变的影响，而慢阶段（F -> O转变）则被强烈减速。在3e（-）还原突变体中，血红素a由于中点电位降低而被氧化，而Cu-A和双核位点被还原。在这种情况下，与O-2的反应通过P-M态进行，因为电子从CuA转移到双核位点被延迟了。在3e-还原突变酶中，膜电位产生的单相，因此对应于P-M -> F转变，是减速的，但其幅度与P-R -> F转变相当。
Arginine 54 in subunit I of cytochrome c oxidase from Paracoccus denitrificans interacts with the formyl group of heme a. Mutation of this arginine to methionine (R54M) dramatically changes the spectral properties of heme a and lowers its midpoint redox potential [Kannt et al, (1999) J. Biol. Chem. 274, 37974-37981; Lee et al, (2000) Biochemistry 39, 2989-2996; Riistama et al. (2000) Biochim. Biophys. Acta 1456, 1-4]. During anaerobic reduction of the mutant enzyme, a small fraction of heme a is reduced first along with heme a(3), while most of heme a is reduced later. This suggests that electron transfer is impaired thermodynamically due to the low redox potential of heme a but that it still takes place from CUA via heme a to the binuclear site as in wild-type enzyme, with no detectable bypass from Cu-A directly to the binuclear site. Consistent with this, the proton translocation efficiency is unaffected at 1 H+/e(-) in the mutant enzyme, although turnover is strongly inhibited. Time-resolved electrometry shows that when the fully reduced enzyme reacts with O-2, the fast phase of membrane potential generation during the PR --> F transition is unaffected by the mutation, whereas the slow phase (F --> O transition) is strongly decelerated. In the 3e(-)-reduced mutant enzyme heme a remains oxidized due to its lowered midpoint potential, whereas Cu-A and the binuclear site are reduced. In this case the reaction with O-2 proceeds via the P-M state because transfer of the electron from CuA to the binuclear site is delayed. The single phase of membrane potential generation in the 3e--reduced mutant enzyme, which thus corresponds to the P-M --> F transition, is decelerated, but its amplitude is comparable to that of the P-R --> F transition.