Date of Award:

5-2009

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Chemistry and Biochemistry

Committee Chair(s)

Lance C. Seefeldt

Committee

Lance C. Seefeldt

Committee

Scott E. Ensign

Committee

Joan Hevel

Abstract

Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown.

By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, could account for the differences in catalytic properties depicted by these enzymes.

The quaternary structure of the dinitrogenases also differs. Such component in the Mo-nitrogenase is an α2β2 tetramer while for the V- an Fe-only nitrogenase is an α2β2δ2 hexamer. The latter enzymes are unable to reduce N2 in the absence of a functional δ subunit, yet they reduce H+ and the non-physiological substrate C2H2. Therefore, the δ subunit is essential for V- and Fe-only dependent nitrogen fixation by a mechanism that still remains unknown. In attempt to understand why the δ subunit is essential for V-dependent N2 reduction from a structural stand point, this work presents the strategy followed to clone the vnfG gene and purify its expression product, the δ subunit. The purified protein was subjected to crystallization trials and used to stabilize a histidine-tagged VFe protein that would otherwise purify with low Fe2+ content and poor H+ and C2H2 reduction activities. The VFe preparation was used to conduct substrate reduction assays to assess: i) The electron allocation patterns to each of the reduction products of the substrates C2H2, N2, N2H4, and N3−; and ii) Inhibition patterns among substrate and inhibitor of the nitrogenase reaction. This work also reports on the effect N2H4 and N3− has on the electron flux to the products of the C2H2 reduction.

The work presented herein provides information with which to compare and contrast biological nitrogen fixation as catalyzed by the Mo- and V-nitrogenases from Azotobacter vinelandii.

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