X-Ray Absorption and Electron Paramagnetic Resonance Studies of Azotobacter Vinelandii Nitrogenase.

Fe K-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used to investigate the metal-cluster structure of both the Mo-containing (Av1) and V-containing (Av1$\sp\prime$) nitrogenase component 1 proteins of Azotobacter vinelandii in their as-isolated and thionine-oxidized states. Using the EXAFS data, plausible distance assignments were made for the cofactor clusters. It was shown that small but consistent distance changes occur in both Av1 and Av1$\sp\prime$ upon thionine oxidation. Furthermore, the EXAFS of Av1$\sp\prime$ is very similar to that of Av1, supporting the proposal that the two proteins have homologous metal cluster structures. Fe K-edge and Mo K-edge EXAFS were used to examine various spectroscopically definable redox states of Av1. It was found that the average metal-metal distances contracted somewhat upon reduction. Also, a split-shell EXAFS analysis was conducted in an attempt to resolve P-cluster and M-center Fe-Fe interactions. Electron Paramagnetic Resonance (EPR) studies of thionine-titrated Av1 were conducted which verified the existence of S = 1/2 and proposed S = 5/2 EPR signals assigned to the singly-oxidized P-cluster, or P$\sp+$. A probabilistic theoretical model was proposed to explain P-cluster oxidation behavior in the $\rm P \to 2\sp{2+}$ range. Oxidative titrations were performed on enzymatically reduced Av1$\sp\prime$ and monitored by EPR spectroscopy. The oxidation behavior of Av1$\sp\prime$ is similar, but not identical to that of Av1, in that it follows a consecutive rather than a random model for the $\rm P\sp+ \to P\sp{2+}$ oxidation. Also, a recently characterized Av1$\sp\prime$ variant called Av1$\sp\prime\sb{\rm A}$ exhibited unique behavior during oxidative titrations. We believe we observe EPR spectral evidence of a redox-gated electron transfer between the P-cluster and the M-center