The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer
The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer
复制标题
DOI：
10.1073/pnas.94.17.9085
复制
发表时间：
1997-08-19
影响因子：
11.1
通讯作者：
Gennis, RB
中科院分区：
文献类型：
--
作者：
Konstantinov, AA;Siletsky, S;Gennis, RB
来自牛和反硝化副球菌的细胞色素c氧化酶的晶体结构揭示了两个假定的质子输入通道，它们连接血红素-铜中心，在那里双氧被还原到内部水相。在这项工作中，我们研究了这两个通道的作用，观察了在球形红杆菌的细胞色素c氧化酶的每个通道中观察到的位点定向突变的影响。利用光电技术监测了与铁氧血红素A (3) （Fe4+ = O2-）光诱导还原为氧化形式（Fe3+OH-）相关的时间分辨率电致质子转移步骤，该氧化还原步骤需要传递“化学”H+来使被还原的氧原子质子化，并且还与质子泵浦相耦合。研究发现，K通道（K362M和T359A）的突变对铁氧到氧化（f到ox）的转变几乎没有影响。虽然稳定状态的周转是严重有限的。相反，这一步骤的电致质子转移受到D通道突变的强烈抑制。结果强烈表明，这两个通道的功能作用不是像最近提出的那样单独递送化学质子或泵送质子[Iwata， S., Ostermeier， C., Ludwig， B. & Michel， H, (1995) Nature (London) 376, 660-669]， D通道可能参与F-to-Ox转变过程中“化学”和“泵送”质子的摄取。而K通道在这部分反应中可能是空的，可能在催化循环的一些早期步骤中用于将质子装载酶，这一结论与K362M和E286Q突变体在有氧稳态周转条件下血红素a(3)的不同氧化还原状态一致。
The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase, In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a(3) (Fe4+ = O2-) to the oxidized form (Fe3+OH-), This redox step requires the delivery of a ''chemical'' H+ to protonate the reduced oxygen atom and is also coupled to proton pumping, It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel, The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H, (1995) Nature (London) 376, 660-669], The D channel is likely to be involved in the uptake of both ''chemical'' and ''pumped'' protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle, This conclusion agrees with different redox states of heme a(3) in the K362M and E286Q mutants under aerobic steady-state turnover conditions.