title: Is Mo involved in hydride binding by the four-electron reduced (E4) intermediate of the nitrogenase MoFe protein?
abstract: We here report the first direct evidence addressing the possible involvement of Mo in substrate interactions during catalytic turnover. When the alpha-70(Ile) MoFe protein is freeze-trapped during H(+) reduction under Ar, the majority of the resting state EPR signal from the molybdenum-iron cofactor (FeMo-co) disappears and is replaced by the S = 1/2 signal of an intermediate that has been shown to be the E(4) MoFe state, which is activated for N(2) binding and reduction through the accumulation of 4 electrons/protons by FeMo-co. ENDOR studies of E(4) showed that it contains two hydrides bound to FeMo-co. We calculate that Mo involvement in hydride binding would require a vector-coupling coefficient for Mo of |K(Mo)| approximately > 0.2 and determine K(Mo) for the E(4) intermediate state through 35 GHz ENDOR measurements of a (95)Mo enriched MoFe protein, further comparing the results with those for the E(0) resting state. The experiments show that Mo of the resting-state FeMo-co is perturbed by the alpha-70(Ile) substitution and that the isotropic (95)Mo hyperfine coupling in E(4) is a(iso) approximately 4 MHz, less than that for the resting state. The decrease in a(iso) for (95)Mo of E(4) from the already small value in the resting state MoFe protein strongly suggests that the resting Mo(IV) is not one-electron reduced during the accumulation of the four electrons of E(4). In any case, the effective K for Mo is very small; |K(Mo)| approximately < 0.04, at least 5-fold less than the lower bound required for Mo to be involved in forming a Mo-H-Fe, hydride. As the hydride couplings also are both far too small and of the wrong symmetry to be associated with a terminal hydride on Mo, we may thus conclude that Mo does not participate in binding a hydride of the catalytically central E(4) intermediate and that only Fe ions are involved. Nonetheless, the response of the Mo coupling to subtle conformational changes in E(0) and to the formation of E(4) suggests that Mo is intimately involved in tuning the geometric and electronic properties of FeMo-co in these states.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukoyanov
authors.forename: Zhi-Yong
authors.initials: ZY
authors.lastname: Yang
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D016948:Azotobacter vinelandii / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry / Q000378:metabolism*; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D008983:Molybdoferredoxin; D008982:Molybdenum