Oxidative Titration of the Nitrogenase VFe Protein from Azotobacter vinelandii: An Example of Redox-Gated Electron Flow

Abstract

The nitrogenase VFe protein of Azotobacter vinelandii (Av1‘) has been shown to exist in two forms called Av1‘_A, which has a primary αβ₂ trimeric structure, and Av1‘_B, which has an α₂β₂ tetrameric structure [Blanchard, C. Z., & Hales, B. J. (1996) Biochemistry 35, 472−478]. Both forms exhibit S = 5/2 EPR signals in the as-isolated state that may be assigned to 1-equiv-oxidized P clusters (P⁺). These signals are abolished by enzymatic reduction with the component 2 protein (Av2‘). Stepwise oxidative titrations of enzymatically reduced Av1‘_B result in the restoration of the S = 5/2 P⁺ signals and the concurrent decrease of the S = 3/2 vanadium cofactor signal. Further oxidation results in the appearance of an integer spin signal assigned to the 2-equiv−oxidized P cluster (P²⁺). Unlike the analogous signal previously observed in Mo nitrogenase component 1 (Av1), which arises from an excited state, the integer spin P²⁺ signal in Av1‘_B originates from a ground-state doublet. Similar oxidative titrations of enzymatically reduced Av1‘_A show redox behavior dramatically different from that of Av1‘_B, as monitored by EPR spectroscopy. We observe spectral evidence for a redox-induced intramolecular electron transfer between the reduced P cluster and the oxidized FeV cofactor cluster during the titrations.