OXIDATIVE TITRATION OF THE NITROGENASE VFE PROTEIN FROM AZOTOBACTER VINELANDII - AN EXAMPLE OF REDOX-GATED ELECTRON FLOW

摘要

The nitrogenase VFe protein of Azotobacter vinelandii (Av1') has been shown to exist in two forms called Av1'(A), which has a primary alpha beta(2) trimeric structure, and Av1'(B), which has an alpha(2) beta(2) tetrameric structure [Blanchard, C. Z., & Hales, B. J. (1996) Biochemistry 35, 372-478]. 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 (p(2+)). 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.