57Fe ENDOR Spectroscopy and 鈥楨lectron Inventory鈥?Analysis of the Nitrogenase E4 Intermediate Suggest the Metal-Ion Core of FeMo-Cofactor Cycles Through Only One Redox Couple

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• 作者：Peter E. Doan ; Joshua Telser ; Brett M. Barney ; Robert Y. Igarashi ; Dennis R. Dean ; Lance C. Seefeldt ; Brian M. Hoffman
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：November 2, 2011
• 年：2011
• 卷：133
• 期：43
• 页码：17329-17340
• 全文大小：1104K
• 年卷期：v.133,no.43(November 2, 2011)
• ISSN：1520-5126

文摘

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 伪-70^Val鈫扞le MoFe protein variant through use of advanced ENDOR methods: 鈥榬andom-hop鈥?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 E₄. In light of the previous findings that FeMo-co of E₄ binds two hydrides in the form of (Fe-(渭-H^{鈥?/sup>)-Fe) fragments, and that molybdenum has not become reduced, an 鈥榚lectron inventory鈥?analysis assigns the formal redox level of FeMo-co metal ions in E₄ to that of the resting state (MN), 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 H₂, the inorganic core of FeMo-co cycles through only a single redox couple connecting two formal redox levels: those associated with the resting state, MN, and with the one-electron reduced state, MR. We further note that this conjecture might apply to other complex FeS enzymes.}