Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Brian J. Hales

Abstract

When Q-Sepharose was used in the purification of the V-nitrogenase proteins from Azotobacter vinelandii, an increase in resolution was observed which resulted in a separation of the nitrogenase component 1 protein (Av1$\sp\prime$) into two forms, labeled Av1$\sp\prime\sb{\rm A}$ and Av1$\sp\prime\sb{\rm B}.$ Even though both forms possessed the same enzymatic behavior, Av1$\sp\prime\sb{\rm A}$ exhibited a lower specific activity and migrated during gel filtration with an apparent lower molecular weight than Av1$\sp\prime\sb{\rm B}.$ Furthermore, SDS-polyacrylamide gel electrophoresis showed different relative compositions of the two major subunits of both forms with Av1$\sp\prime\sb{\rm A}$ possessing a trimer $(\alpha\beta\sb2)$ pattern compared to the tetramer $(\alpha\sb\beta\sb2)$ pattern found for Av1$\sp\prime\sb{\rm B}.$ Metal analysis indicated a V to Fe ratio of 1:19 for Av1$\sp\prime\sb{\rm A}$ and 2:30 for Av1$\sp\prime\sb{\rm B}.$ EPR spectroscopy revealed that both proteins retained the S = 3/2 and S = 1/2 signals observed in earlier isolations with an additional S = 1/2 signal present in the spectrum of protein A. These results suggest that AV1$\sp\prime\sb{\rm A}$ is an incomplete form of the VFe protein, containing only one cofactor and one P-cluster with an additional (Fe$\sb4$-S$\sb4$) cluster. The presence of a V-storage protein in A. vinelandii was also investigated. Although no V-storage protein was found, two V-binding proteins were observed. Inactivation of V- and Mo-nitrogenase by organic solvents was investigated by kinetic measurements. The dependence of catalytic activities on cosolvent concentration resulted in a sigmoidal shape curve for most systems studied, which indicated cooperative type interactions. Ethanol demonstrated noncooperative inhibition of V-nitrogenase. Ethylene glycol and NaCl have been shown to affect the enzyme by similar mechanisms in which relaxation state of component 1 protein, association of the nitrogenase proteins, and/or binding of MgATP to the Fe-protein are believed to be affected. These cosolvents provide an alternative means for quenching the nitrogenase reaction. The Arrhenius plot for V-nitrogenase is biphasic with a break at 25$\sp\circ$C. Activation energies were calculated to be 22 and 82 kJ/mol above and below the break, respectively. The addition of ethylene glycol was shown to produced a higher activation energy at lower temperatures.

Pages

109

DOI

10.31390/gradschool_disstheses.5998

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