title: Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme.
abstract: We proposed a reductive elimination/oxidative addition (re/oa) mechanism for reduction of N2 to 2NH3 by nitrogenase, based on identification of a freeze-trapped intermediate of the α-70(Val→Ile) MoFe protein as the Janus intermediate that stores four reducing equivalents on FeMo-co as two [Fe-H-Fe] bridging hydrides (denoted E4(4H)). The mechanism postulates that obligatory re of the hydrides as H2 drives reduction of N2 to a state (denoted E4(2N2H)) with a moiety at the diazene (HN═NH) reduction level bound to the catalytic FeMo-co. EPR/ENDOR/photophysical measurements on wild type (WT) MoFe protein now establish this mechanism. They show that a state freeze-trapped during N2 reduction by WT MoFe is the same Janus intermediate, thereby establishing the α-70(Val→Ile) intermediate as a reliable guide to mechanism. Monitoring the Janus state in WT MoFe during N2 reduction under mixed-isotope condition, H2O buffer/D2, and the converse, establishes that the bridging hydrides/deuterides do not exchange with solvent during enzymatic turnover, thereby solving longstanding puzzles. Relaxation of E4(2N2H) to the WT resting-state is shown to occur via oa of H2 and release of N2 to form Janus, followed by sequential release of two H2, demonstrating the kinetic reversibility of the re/oa equilibrium. Relative populations of E4(2N2H)/E4(4H) freeze-trapped during WT turnover furthermore show that the reversible re/oa equilibrium between [E4(4H) + N2] and [E4(2N2H) + H2] is ∼ thermoneutral (ΔreG(0) ∼ -2 kcal/mol), whereas, by itself, hydrogenation of N2(g) is highly endergonic. These findings demonstrate that (i) re/oa accounts for the historical Key Constraints on mechanism, (ii) that Janus is central to N2 reduction by WT enzyme, which (iii) indeed occurs via the re/oa mechanism. Thus, emerges a picture of the central mechanistic steps by which nitrogenase carries out one of the most challenging chemical transformations in biology.
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukoyanov
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
authors.forename: Nimesh
authors.initials: N
authors.lastname: Khadka
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, Virginia Tech , Blacksburg, Virginia 24061, United States.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University , Logan, Utah 84322, United States.
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
authors.affiliation: Departments of Chemistry and Molecular Biosciences, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009584:Nitrogen / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D006859:Hydrogen; D009591:Nitrogenase; D009584:Nitrogen