~(57)Fe ENDOR Spectroscopy and 'Electron Inventory' Analysis of the Nitrogenase E_4 Intermediate Suggest the Metal-Ion Core of FeMo-Cofactor Cycles Through Only One Redox Couple

摘要

N_2 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 (E_3 or E_4 states), with the E_4 state being optimally activated. Here we study the FeMo-co ~(57)Fe atoms of E_4 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 ~(57)Fe hyperfine couplings for five of the seven iron sites of the reductively activated E_4 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 ~(57)Fe 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 E_4 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 E_4 to that of the resting state (M~N), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier ~(57)Fe 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 N_2 plus 2H~+ to two NH~3 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 . We further note that this conjecture might apply to other complex FeS enzymes.