Is Mo involved in hydride binding by the four-electron reduced (E ₄) intermediate of the nitrogenase MoFe protein?

(Figure Presented) We here report the first direct evidence addressing the possible involvement of Mo in substrate interactions during catalytic turnover. When the α-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 E₄ MoFe state, which is activated for N₂ binding and reduction through the accumulation of 4 electrons/protons by FeMo-co. ENDOR studies of E₄ 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| > 0.2 and determine K_Mo for the E ₄ intermediate state through 35 GHz ENDOR measurements of a ⁹⁵Mo enriched MoFe protein, further comparing the results with those for the E₀ resting state. The experiments show that Mo of the resting-state FeMo-co is perturbed by the α-70^Ile substitution and that the isotropic ⁹⁵Mo hyperfine coupling in E₄ is a_iso ≈ 4 MHz, less than that for the resting state. The decrease in a_iso for ⁹⁵Mo of E₄ 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 E₄. In any case, the effective K for Mo is very small; |K _Mo|≲ 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 E₄ intermediate and that only Fe ions are involved. Nonetheless, the response of the Mo coupling to subtle conformational changes in E₀ 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.