DETECTION OF EPR SIGNALS ASSIGNED TO THE 1-EQUIV-OXIDIZED P-CLUSTERS OF THE NITROGENASE MOFE-PROTEIN FROM AZOTOBACTER-VINELANDII

Component 1 (the MoFe protein) of conventional nitrogenase from Azotobacter vinelandii contains two types of metal clusters, called M-centers and P-clusters. The M-centers, or FeMo-co, are paramagnetic in the as-isolated form of the protein, exhibiting an EPR signal typical of a rhombic S = 3/2 system. The P-clusters are diamagnetic in the as-isolated form of the protein but become paramagnetic upon chemical or potentiometric oxidation. We have undertaken a controlled oxidative titration of component 1 with thionine solution and observed EPR signals believed to originate from paramagnetic P-clusters. We present EPR spectra associated with half-integer S = 5/2 and S = 1/2 spin states and propose that these spectra arise from the 1-equiv-oxidized form of the P-cluster. Spectral analysis and theoretical models are presented which support the argument that P-cluster oxidation occurs 1 equiv at a time and that the 1-equiv-oxidized P-clusters are stable species in the oxidative titration of Av1.