⁵⁷Fe ENDOR spectroscopy and ?electron inventory? analysis of the nitrogenase E₄ intermediate suggest the metal-ion core of FeMo-cofactor cycles through only one redox couple

N₂ binds to the active-site metal cluster in the nitrogenase MoFe protein, the FeMo-cofactor ([7Fe-9S-Mo-homocitrate-X]; FeMo-co) only after the MoFe protein has accumulated three or four electrons/protons (E₃ or E₄ states), with the E₄ state being optimally activated. Here we study the FeMo-co ⁵⁷Fe atoms of E₄ trapped with the α-70^Val→Ile MoFe protein variant through use of advanced ENDOR methods: ?random-hop? Davies pulsed 35 GHz ENDOR; difference triple resonance; the recently developed Pulse-Endor-SaTuration and REcovery (PESTRE) protocol for determining hyperfine-coupling signs; and Raw-DATA (RD)-PESTRE, a PESTRE variant that gives a continuous sign readout over a selected radiofrequency range. These methods have allowed experimental determination of the signed isotropic ⁵⁷Fe hyperfine couplings for five of the seven iron sites of the reductively activated E₄ FeMo-co, and given the magnitude of the coupling for a sixth. When supplemented by the use of sum-rules developed to describe electron-spin coupling in FeS proteins, these ⁵⁷Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E ₄. In light of the previous findings that FeMo-co of E₄ binds two hydrides in the form of (Fe-(μ-H^-)-Fe) fragments, and that molybdenum has not become reduced, an ?electron inventory? analysis assigns the formal redox level of FeMo-co metal ions in E₄ to that of the resting state (M^N), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier ⁵⁷Fe ENDOR studies and electron inventory analyses of the bio-organometallic intermediate formed during the reduction of alkynes and the CO-inhibited forms of nitrogenase (hi-CO and lo-CO) inspire the conjecture that throughout the eight-electron reduction of N₂ plus 2H⁺ to two NH₃ plus H ₂, the inorganic core of FeMo-co cycles through only a single redox couple connecting two formal redox levels: those associated with the resting state, M^N, and with the one-electron reduced state, M^R. We further note that this conjecture might apply to other complex FeS enzymes.