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 vertical bar K-Mo vertical bar greater than or similar to 0.2 and determine K-Mo for the E-4 intermediate state through 35 GHz ENDOR measurements of a Mo-95 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 Mo-95 hyperfine Coupling in E-4 is a(iso) approximate to 4 MHz, less than that for the resting state. The decrease in a(iso) for Mo-95 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; vertical bar K-Mo vertical bar less than or similar to 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.