Inhibition of proton transfer in cytochrome c oxidase by zinc ions:: delayed proton uptake during oxygen reduction
Inhibition of proton transfer in cytochrome c oxidase by zinc ions:: delayed proton uptake during oxygen reduction
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DOI：
10.1016/s0005-2728(02)00268-2
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发表时间：
2002-09-10
影响因子：
4.3
通讯作者：
Brzezinski, P
中科院分区：
文献类型：
--
作者：
Aagaard, A;Namslauer, A;Brzezinski, P
我们研究了锌离子对细胞色素C氧化酶质子转移反应的影响。在没有锌离子的情况下，从“过氧基”(P-R)到“铁基”(F)中间体的转变的时间常数接近100微秒，它与质子从本体溶液中的转移有关，具有F转变的本征时间常数。而在100微米的锌离子存在下，P-R-->；F转变的速度减慢了与2相似的倍数，而从主体溶液中吸收质子的能力受到了更大程度的损害。相反，在下一个反应步骤中，大约有两个质子(一个在没有锌离子的情况下，一个质子)被占用，即F衰变为氧化的(O)酶，其时间常数类似于2.5ms。因此，结果表明，酶中有一个质子可用于氧还原，并证实了我们之前的观察结果，即F可以在没有从本体溶液中摄取质子的情况下形成。在突变酶中，D途径入口点的天冬氨酸(I-132)被其非质子化类似物Asn取代，对锌离子没有影响。此外，在D2O中测量时，没有观察到Zn2+对F--&Gt；O跃迁速率的影响，因为在D2O中，跃迁在内部被减慢到接近10ms，这已经比结合的Zn2+慢。结合早期的结果表明，P-R-->；F和F-->；O跃迁都与D-途径的质子吸收有关，本研究的结果表明，锌离子与D-途径结合并阻断了D-途径的入口。(C)2002 Elsevier Science B.V.保留所有权利。
We have investigated the effect of Zn ions on proton-transfer reactions in cytochrome c oxidase. In the absence of Zn2+ the transition from the "peroxy" (P-R) to the "ferryl" (F) intermediate has a time constant of similar to100 mus and it is associated with proton transfer from the bulk solution with an intrinsic time constant of F transition. While in the presence of 100 muM Zn2+ the P-R-->F transition was slowed by a factor of similar to2, proton uptake from the bulk solution was impaired to a much greater extent. Instead, about two protons (one proton in the absence of Zn2+) were taken up during the next reaction step, i.e. the decay of F to the oxidized (O) enzyme with a time constant of similar to 2.5 ms. Thus, the results show that there is one proton available within the enzyme that can be used for oxygen reduction and confirm our previous observation that F can be formed without proton uptake from the bulk solution. No effect of Zn2+ was observed with a mutant enzyme in which Asp(I-132), at the entry point of the D-pathway, was replaced by its non-protonatable analogue Asn. In addition, no effect of Zn2+ was observed on the F --> O transition rate when measured in D2O, because in D2O, the transition is internally slowed to similar to 10 ms, which is already slower than with bound Zn2+. Together with earlier results showing that both the P-R --> F and F --> O transitions are associated with proton uptake through the D-pathway, the results from this study indicate that Zn2+ binds to and blocks the entrance of the D-pathway. (C) 2002 Elsevier Science B.V. All rights reserved.