Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex

Nitrogenases are metalloenzymes that catalyse the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle^1,2. Although bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases have remained unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a cryo-electron microscopy structure of a native nitrogenase-PII protein supercomplex from Methanosarcina acetivorans. The structure reveals an assembly of three NifDK heterotetramers bridged by six NifI_1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The PII complexes show asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro. This higher-order architecture identifies a regulatory strategy in methanogens in which PII proteins drive nitrogenase oligomerization to control activity. The finding that nitrogenase activity may be modulated through direct assembly into higher-order structures indicates future directions for the exploration of nitrogenase evolution, regulation and biotechnological applications.