(Figure Presented) We here report the first direct evidence addressing the possible involvement of Mo in substrate interactions during catalytic turnover. When the α-70Ile 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 |KMo| > 0.2 and determine KMo 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-70Ile substitution and that the isotropic 95Mo hyperfine coupling in E4 is aiso ≈ 4 MHz, less than that for the resting state. The decrease in aiso 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.
ASJC Scopus subject areas
- Colloid and Surface Chemistry