Redox-state-dependent CO binding in MoFe nitrogenase: insights from EPR, IR, and QM/MM analysis

Nitrogenase drives biological nitrogen fixation through an eight-step cycle, in which "n" electrons and protons are accumulated across the E _n catalytic states. Carbon monoxide (CO), a noncompetitive inhibitor for Mo nitrogenase but a substrate for V- or Fe-only nitrogenases, has been widely used as a spectroscopic probe of nitrogenase's active site. Infrared (IR) spectroscopy reports on CO binding mode and coordination environments, while electron paramagnetic resonance (EPR) spectroscopy characterizes CO-bound states via their non-integer spin states. Crystal structures reveal a µ₂-bridging mode formed by substitution of a "belt" sulfide, suggesting that sulfide lability may be mechanistically relevant. However, the redox state at which CO binds and the assignment of the resulting non-integer spin states remain unresolved, obscuring their relationship to the N₂ reduction cycle. Likewise, the mechanism and relevance of sulfide substitution remain unclear. Here, we combine static and stopped-flow IR, EPR spectroscopy, photolysis, and quantum mechanical/molecular mechanical (QM/MM) calculations, supported by non-negative matrix factorization (NMF), to investigate CO binding under varying electron flux, varying CO pressure, and H₂ pressure. We find that CO binds predominantly at the E₁ state, whereas EPR-active species correspond to CO-bound E₂ states. H₂S is identified as the preferred leaving group, and the absence of hydrides supports a stable CO-bound state that is disconnected from the reductive steps of nitrogen fixation.