Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Brian J. Hales

Abstract

Electron Paramagnetic Resonance (EPR) spectroscopy was used to examine the paramagnetism of the clusters in nitrogenase from Azotobacter vinelandii. The EPR spectrum of nitrogenase (Av1 and Av2) was examined in the presence of an extrinsic paramagnet, DyEDTA, at different concentrations of NaCl, during thionine oxidation (Av1), and in the presence of MgATP (Av2). Cross combinations of Av1 and Av2 were also investigated in the presence and absence of the chemical cross-linker, EDC as well as with the nitrogenase components from Clostridium pasteurianum. From relaxation enhancement measurements, the distance of closest approach of DyEDTA to the intrinsic metal clusters in Av1 and Av2 was calculated. For Av2, the distance was calculated to be 5 A. The calculated distance for Av1, however, put the M center practically on the surface of the protein. One reason for this (probably) spurious result may be that the distance calculation is derived for a model with total spin S = 1/2. The M center in Av1 is a faster-relaxing S = 3/2 system which will affect the different terms of the dipolar Hamiltonian in ways different from an S = 1/2 system. The results of the thionine oxidation of Av1 provided evidence of dipolar interactions between paramagnetic P clusters and the paramagnetic M centers. These results were fitted to a model for the interaction and arrangement of the P clusters relative to the M centers. The paramagnetism of the metal clusters of Av1$\sp\prime$ (the alternative nitrogenase from Azotobacter vinelandii) was investigated as a function of (1) enzymatic turnover; (2) thionine oxidation of Av1$\sp\prime$; (3) the effects of pH on Av1$\sp\prime$; and (4) isolation of the cofactor. The S = 1/2 signal was completely quenched upon completion of turnover, while the S = 3/2 signal decreased only slightly in intensity. Thionine oxidation of Av1$\sp\prime$ produced analogous results in which the S = 1/2 signal was oxidized with one-half equivalent of thionine. The S = 3/2 signal required almost four equivalents before decreasing significantly in amplitude. Increasing the pH of Av1$\sp\prime$ shifted the S = 3/2 signal and the S = 1/2 signal increased in amplitude. Finally, FeVco, the cofactor or M center of Av1$\sp\prime$, was successfully extracted into DMF but not into NMF.

Pages

225

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