title: 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.
abstract: 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(R). We further note that this conjecture might apply to other complex FeS enzymes.
authors: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors: Peter E
authors: PE
authors: Doan
authors: Joshua
authors: J
authors: Telser
authors: Brett M
authors: BM
authors: Barney
authors: Robert Y
authors: RY
authors: Igarashi
authors: Dennis R
authors: DR
authors: Dean
authors: Lance C
authors: LC
authors: Seefeldt
authors: Brian M
authors: BM
authors: Hoffman
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D007503:Iron Isotopes; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D007503:Iron Isotopes; D008983:Molybdoferredoxin; D009591:Nitrogenase