Structures of protein-protein complexes involved in electron transfer.
Structures of protein-protein complexes involved in electron transfer.
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电子转移 (ET) 反应对于生命至关重要，因为它们支持氧化磷酸化和光合作用（导致 ATP 生成的过程），并参与中间代谢的许多反应。这些作用的关键是瞬时蛋白质间 ET 复合物的形成。由于这些弱瞬时复合物在晶体学研究中仍然很难处理，因此缺乏控制伴侣蛋白之间特异性的结构基础。蛋白质间 ET 过程是反硝化所有关键步骤的核心，反硝化是一种替代呼吸形式，当氧气浓度有限时，细菌通过气态中间体一氧化氮 (NO) 和一氧化二氮 (N2O) 将硝酸盐或亚硝酸盐还原为 N2。亚硝酸盐单电子还原为 NO（N2O 的前体）是由含血红素或含铜的亚硝酸盐还原酶 (CuNiR) 进行的，其中它们从氧化还原伙伴蛋白（铜氧还蛋白或 c 型细胞色素）接收电子。在这里，我们以 1.01Å 分辨率报道了来自 Ralstonia pickettii (RpNiR) 的新表征的三结构域 hemec-Cu 亚硝酸还原酶及其 M92A 和 P93A 突变体的结构。非常高分辨率提供了 CuNiR 的核心三聚铜氧还蛋白结构和束缚的细胞色素 c 结构域之间界面的原子细节的第一视图，该结构允许酶充当有效的自电子转移系统，即供体和受体蛋白通过基因组采集融合在一起以获得功能优势。 RpNiR 与 CuNiR 与供体蛋白 AxNiR-cytc551 的二元复合物的比较以及诱变研究为 ET 界面处氢键水的重要性提供了直接证据。该结构还解释了亚硝酸盐优先与 RpNiR 活性位点处的还原铜离子结合，与其他 CuNiR 相比，其他 CuNiR 会发生还原失活，从而阻止底物结合。
Electron transfer (ET) reactions are essential for life since they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism. Key to these roles is the formation of transient inter-protein ET complexes. The structural basis for the control of specificity between partner proteins is lacking since these weak transient complexes have remained largely intractable for crystallographic studies. Inter-protein ET processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 via the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one electron reduction of nitrite to NO, a precursor to N2O, is performed by either a heme- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome. Here we report the structures of the newly characterized three-domain hemec-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01Å resolution and its M92A and P93A mutains. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system i.e. where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551, and mutagenesis studies provide direct evidence for the importance of a hydrogen bonded water at the interface in ET. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.