Addison Holly, Pfister Pascal, Lago-Maciel Ana, Erb Tobias J, Pierik Antonio J, Rebelein Johannes G
Microbial Metalloenzymes Research Group, Max Planck Institute for Terrestrial Microbiology, Marburg, 35043, Germany.
Biochemistry and Synthetic Metabolism Research Group, Max Planck Institute for Terrestrial Microbiology, Marburg, 35043, Germany.
Chemistry. 2025 Jul 2;31(37):e202500844. doi: 10.1002/chem.202500844. Epub 2025 May 30.
Ferredoxins deliver electrons to drive many challenging biochemical transformations, including enzyme-catalyzed nitrogen fixation. We recently showed two distinct ferredoxins, FdC and FdN, were essential for iron nitrogenase-mediated nitrogen fixation in R. capsulatus. In this study, we perform investigations on FdC and FdN to establish their key differences in terms of specificity, structure, and electronic properties. In vivo complementation studies of both the genes encoding FdC (fdxC) and FdN (fdxN), into ∆fdxC and ∆fdxN R. capsulatus-deletion strains under N-fixing conditions, showed that plasmid-based expression of fdxN recovered diazotrophic growth and Fe-nitrogenase activity in both ∆fdxC and ∆fdxN strains, while plasmid-based fdxC expression could only complement the ∆fdxC strain. Spectroscopic analysis of FdC and FdN using electron paramagnetic resonance spectroscopy revealed large differences in the electronic features of FdC and FdN. These differences were accompanied by large structural differences between FdC and FdN, assessed by a crystallographic structure of FdC and an AlphaFold model of FdN. We report novel features in the FdC structure, in terms of secondary structure and hydrogen-bonding network, compared with structures of other [FeS]-cluster ferredoxins. Overall, we explore the biophysical properties that influence ferredoxin specificity, while providing new insights into the properties of ferredoxins essential for N-fixation.
铁氧化还原蛋白传递电子以驱动许多具有挑战性的生物化学转化过程，包括酶催化的固氮作用。我们最近发现，两种不同的铁氧化还原蛋白FdC和FdN，对于荚膜红细菌中铁固氮酶介导的固氮作用至关重要。在本研究中，我们对FdC和FdN进行了研究，以确定它们在特异性、结构和电子性质方面的关键差异。在固氮条件下，将编码FdC（fdxC）和FdN（fdxN）的基因进行体内互补研究，导入∆fdxC和∆fdxN荚膜红细菌缺失菌株中，结果表明，基于质粒的fdxN表达恢复了∆fdxC和∆fdxN菌株的固氮生长和铁固氮酶活性，而基于质粒的fdxC表达只能互补∆fdxC菌株。使用电子顺磁共振光谱对FdC和FdN进行光谱分析，揭示了FdC和FdN电子特征的巨大差异。这些差异伴随着FdC和FdN之间的巨大结构差异，这是通过FdC的晶体结构和FdN的AlphaFold模型评估得出的。与其他[FeS]簇铁氧化还原蛋白的结构相比，我们报道了FdC结构在二级结构和氢键网络方面的新特征。总体而言，我们探索了影响铁氧化还原蛋白特异性的生物物理性质，同时为固氮所需的铁氧化还原蛋白的性质提供了新的见解。
