Proton transfer during reduction of the catalytic metallo-cofactors of the three nitrogenase isozymes

Nitrogenase catalyzes biological nitrogen fixation, the conversion of atmospheric N₂ into bioavailable ammonia. The three nitrogenase isozymes-Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase-utilize catalytic cofactors distinguished by their metal composition (Fe₇M, M = Mo, V, or Fe; denoted FeM-co). Their catalytic cycles involve stepwise addition of 8[e^-/H⁺] to FeM-co, generating intermediates designated E _n , where n is the number of [e^-/H⁺] delivered. The electron-transfer has been extensively characterized, but the proton delivery has not. Here, we investigate [e^-/H⁺] delivery during early-stage conversions, primarily E₀ → E₁(H), for each of the three nitrogenases, using as reductants γ-ray-generated thermolyzed, mobile electrons at 77 K, and radiation-generated solvent radicals during subsequent annealing to higher temperatures. Our results show E₀ → E₁(H) conversion differs among the three MFe-proteins. The FeMo-co of MoFe-protein accepts an electron (ET) during 77 K γ-irradiation, but proton transfer (PT) to generate E₁(H) is only enabled by conformational or thermodynamic activation upon cryoannealing to ∼200 K(ET/PT). For VFe-protein, E₁(H) forms during annealing at-and-above 210 K by electron-transfer to FeV-co from radicals through proton-coupled electron transfer (PCET), which too is enabled by activated proton transfer. FeFe-protein differs in directly exhibiting delivery of protons at 77 K, which together with the mobile electrons react to form E₁(H). This could well occur by PCET at 77 K, but does not preclude the possibility of sequential 77 K electron/proton transfer (ET/PT). In addition, 450 nm photolysis reveals the E₁(H) state of FeV-co, like that of FeFe-co, contains a hydride bound to a formally oxidized cofactor. The mechanistic differences observed here provide a contribution towards understanding the sources of catalytic differences among the three nitrogenase isozymes.