Jasaitis A, Backgren C, Morgan J E, Puustinen A, Verkhovsky M I, Wikström M
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, University of Helsinki, Finland.
Biochemistry. 2001 May 1;40(17):5269-74. doi: 10.1021/bi002948b.
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 Cu(A) 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 P(R )()--> 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 Cu(A) 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. From this we conclude that the completely (4e(-)) reduced enzyme is fully capable of proton translocation.
反硝化副球菌细胞色素c氧化酶亚基I中的精氨酸54与血红素a的甲酰基相互作用。将该精氨酸突变为甲硫氨酸（R54M）会显著改变血红素a的光谱特性并降低其中点氧化还原电位[坎特等人（1999年）《生物化学杂志》274卷，37974 - 37981页；李等人（2000年）《生物化学》39卷，2989 - 2996页；里斯塔马等人（2000年）《生物化学与生物物理学报》1456卷，1 - 4页]。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a3一起被还原，而大部分血红素a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损害，但它仍然像野生型酶一样从铜（A）经由血红素a发生到双核位点，没有检测到从铜（A）直接到双核位点的旁路。与此一致的是，尽管周转受到强烈抑制，但突变酶的质子转运效率在1 H⁺/e⁻时不受影响。时间分辨电位测定表明，当完全还原的酶与O₂反应时，在P（R）→F转变期间膜电位产生的快速相不受突变影响，而慢速相（F→O转变）则强烈减速。在3e⁻还原的突变酶中，由于其降低的中点电位，血红素a保持氧化状态，而铜（A）和双核位点被还原。在这种情况下，与O₂的反应通过P（M）状态进行，因为从铜（A）到双核位点的电子转移被延迟。3e⁻还原的突变酶中膜电位产生的单相，即对应于P（M）→F转变，被减速，但其幅度与P（R）→F转变的幅度相当。由此我们得出结论，完全（4e⁻）还原的酶完全能够进行质子转运。
