All nitrogen-fixing bacteria and archaea utilize a unique series of enzymes with complex metal cofactors to convert atmospheric dinitrogen into ammonia. These enzymes, collectively known as nitrogenases, are classified based on their metal cofactors into molybdenum (Mo), vanadium (V), or iron-only (Fe) nitrogenases. Nitrogenase consists of two metalloprotein components: the iron (Fe) protein (NifH) and the molybdenum-iron (MoFe) protein (NifDK) or its homologous counterparts. Structurally, the MoFe protein exist as an α2β2 tetramer, while the Fe protein exists as a homodimer harboring a [4Fe-4S] cluster. Electron transfer occurs through a stepwise relay from the Fe protein to the MoFe protein, involving the P-cluster and the catalytic FeMo cofactor.
Despite extensive studies on nitrogenase, key aspects of its regulation remain enigmatic, particularly in methanogenic archaea where unique inhibitory mechanisms may influence its function. Here, we employed a combination of structural and biochemical approaches to probe nitrogenase under conditions that mimic its native regulatory environment. Our cryo-EM findings unveil distinct protein-mediated interactions that modulate nitrogenase activity through a hierarchical and coordinated inhibition process. The interplay of these regulatory elements suggests an intricate network of structural complexity and inhibitory mechanism, potentially involving conformational gating and higher order complex structure within the catalytic core.
The insights gained from this study provide a deeper understanding of nitrogenase regulation in methanogens, distinguishing it from its bacterial counterparts. These findings highlight the complexity of enzymatic control in anaerobic archaea and pave the way for future investigations into nitrogen fixation strategies and their evolutionary implications.