The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase.

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad = 3/2 EPR signal (Kramers state) having values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V, 3Fe, 4Fe]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, = 0, or non-Kramers, integer-spin ( = 1, 2 ). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic = 1/2 EPR signal from such a reduced state is observed, with = [2.18, 2.12, 2.09] and showing well-defined V ( = 7/2) hyperfine splitting, = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V, 4Fe, 3Fe]. Our findings suggest that the V does not change valency throughout the catalytic cycle.