Microbial Metabolism Research Group, Max Planck Institute for Marine Microbiology, Bremen, Germany.
Department of Inorganic and Analytical Chemistry, Faculty of Sciences, University of Geneva, Switzerland.
FEBS J. 2024 Aug;291(15):3454-3480. doi: 10.1111/febs.17148. Epub 2024 May 2.
The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C. Here, we structurally and biochemically analyse NifHs from three Methanococcales, offering the X-ray crystal structures from meso-, thermo-, and hyperthermophilic methanogens. While NifH from Methanococcus maripaludis (37 °C) was obtained through heterologous recombinant expression, the proteins from Methanothermococcus thermolithotrophicus (65 °C) and Methanocaldococcus infernus (85 °C) were natively purified from the diazotrophic archaea. The structures from M. thermolithotrophicus crystallised as isolated exhibit high flexibility. In contrast, the complexes of NifH with MgADP obtained from the three methanogens are superposable, more rigid, and present remarkable structural conservation with their homologues. They retain key structural features of P-loop NTPases and share similar electrostatic profiles with the counterpart from the bacterial model organism Azotobacter vinelandii. In comparison to the NifH from the phylogenetically distant Methanosarcina acetivorans, these reductases do not cross-react significantly with Mo-nitrogenase from A. vinelandii. However, they associate with bacterial nitrogenase when ADP· is added to mimic a transient reactive state. Accordingly, detailed surface analyses suggest that subtle substitutions would affect optimal binding during the catalytic cycle between the NifH from Methanococcales and the bacterial nitrogenase, implying differences in the N-machinery from these ancient archaea.
氮酶还原酶 NifH 催化 ATP 依赖性电子向 Mo-氮酶的传递，该反应是生物固氮的核心。尽管已经对细菌中的 NifH 进行了广泛研究，但有关其古菌对应物的结构信息有限。古菌的 NifH 被认为更为古老，特别是来自 Methanococcales 的 NifH，这是一群海洋产氢甲烷菌，其中包括在接近 92°C 的温度下生长的固氮生物。在这里，我们对来自三个 Methanococcales 的 NifH 进行了结构和生化分析，提供了从中温、高温和超高温甲烷菌获得的 X 射线晶体结构。Methanococcus maripaludis（37°C）的 NifH 是通过异源重组表达获得的，而 Methanothermococcus thermolithotrophicus（65°C）和 Methanocaldococcus infernus（85°C）的蛋白则是从固氮古菌中天然纯化的。M. thermolithotrophicus 的结构在孤立状态下结晶时表现出高度的灵活性。相比之下，从三种甲烷菌获得的 NifH 与 MgADP 的复合物是可叠加的，更刚性，并且与它们的同源物具有显著的结构保守性。它们保留了 P 环 NTP 酶的关键结构特征，并与细菌模式生物 Azotobacter vinelandii 的对应物具有相似的静电分布。与系统发育上遥远的 Methanosarcina acetivorans 的 NifH 相比，这些还原酶与 A. vinelandii 的 Mo-氮酶没有明显的交叉反应。然而，当添加 ADP· 以模拟瞬态反应状态时，它们会与细菌氮酶结合。因此，详细的表面分析表明，细微的取代会影响 Methanococcales 的 NifH 与细菌氮酶之间催化循环中的最佳结合，这表明这些古老古菌的 N 机制存在差异。
