EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ
EPR characterization of ubisemiquinones and iron-sulfur cluster N2, central components of the energy coupling in the NADH-ubiquinone oxidoreductase (complex I) in situ
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DOI：
10.1023/a:1016083419979
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发表时间：
2002-06-01
影响因子：
3
通讯作者：
Ohnishi, T
中科院分区：
文献类型：
--
作者：
Magnitsky, S;Toulokhonova, L;Ohnishi, T
质子转运的NADH-泛醌氧化还原酶（复合物I）是最大的和最不了解的呼吸复合物。内在的氧化还原组分（FMN和铁硫簇）驻留在复合物的海角部分。泛醌是膜部分质子泵送反应中最可能的关键参与者。在这里，我们报告的存在下，三个不同的半醌物种在复杂的I原位，显示出广泛不同的自旋弛豫曲线。作为我们的第一种方法，半醌形式在紧密耦合的激活的牛心亚线粒体颗粒中的稳态NADH-泛醌-1（Q（1））反应期间被捕获，并被命名为SQ（Nf）（快速松弛组分）、SQ（N）s（缓慢松弛组分）和SQ（Nx）（非常缓慢松弛组分）。这表明在复合物I中存在至少三个不同的醌结合位点。在目前的研究中，特别注意的是放在SQ（N）f，因为它的高灵敏度的三角洲（亩）的代（H+）和特定的复合物I抑制剂（鱼藤酮和piericidin A）以独特的方式。鱼藤酮抑制正向电子转移反应比抑制反向反应更强烈，而粉蝶啶A以类似的效力抑制两种反应。鱼藤酮以比淬灭较慢松弛组分（SQ（Ns）和SQ（Nx））所需的浓度低得多的浓度淬灭SQ（Nf）信号。结果表明，G的线形变化与G的线形变化之间存在密切的相关性。（平行于）= 2.05的簇N2信号和SQ（N）f信号的猝灭，使用两种不同的实验方法：（1）通过寡霉素滴定改变Delta（μ）对波浪线（H+）泊，这减少了穿过SMP膜的质子泄漏;（2）用不同浓度的鱼藤酮抑制反向电子传递。这些新的实验结果进一步加强了我们先前的提议，即SQ（Nf）和团簇N2之间发生了直接的自旋耦合。我们讨论了这些发现的影响，在复杂的能量耦合机制。
The proton-translocating NADH-ubiquinone oxidoreductase (complex I) is the largest and least understood respiratory complex. The intrinsic redox components (FMN and iron-sulfur clusters) reside in the promontory part of the complex. Ubiquinone is the most possible key player in proton-pumping reactions in the membrane part. Here we report the presence of three distinct semiquinone species in complex I in situ, showing widely different spin relaxation profiles. As our first approach, the semiquinone forms were trapped during the steady state NADH-ubiquinone-1 (Q(1)) reactions in the tightly coupled, activated bovine heart submitochondrial particles, and were named SQ(Nf) (fast-relaxing component), SQ(N)s (slow-relaxing), and SQ(Nx) (very slow relaxing). This indicates the presence of at least three different quinone-binding sites in complex I. In the current study, special attention was placed on the SQ(N)f, because of its high sensitivities to Delta(mu) over tilde (H+) and to specific complex I inhibitors (rotenone and piericidin A) in a unique manner. Rotenone inhibits the forward electron transfer reaction more strongly than the reverse reaction, while piericidine A inhibits both reactions with a similar potency. Rotenone quenched the SQ(Nf) signal at a much lower concentration than that required to quench the slower relaxing components (SQ(Ns) and SQ(Nx)). A close correlation was shown between the line shape alteration of the g(parallel to) = 2.05 signal of the cluster N2 and the quenching of the SQ(N)f signal, using two different experimental approaches: (1) changing the Delta(mu) over tilde (H+) poise by the oligomycin titration which decreases proton leak across the SMP membrane; (2) inhibiting the reverse electron transfer with different concentrations of rotenone. These new experimental results further strengthen our earlier proposal that a direct spin-coupling occurs between SQ(Nf) and cluster N2. We discuss the implications of these findings in connection with the energy coupling mechanism in complex I.