Brian.Hales@chemgate.chem.lsu.edu
The research in our laboratory centers on the study of structure/function relationships of the metal centers in various metalloproteins. Currently, our prime focus is in the area of nitrogen fixation. Nitrogen fixation, the enzymatic conversion of dinitrogen into ammonia, N2 + 8H+ + 8e <==> 2NH3 + H2, is directly related to worldwide crop production and, as such, is one of the most important biological reactions in nature. Obviously, from the point of view of agriculture alone, it would be of great importance to have a clear understanding of the enzymology of this reaction. However, in spite of the large volume of research on nitrogen fixation during the past 30 years, the mechanism of this reaction is still unclear.
What is known is that the nitrogen-fixing enzyme, nitrogenase, is synthesized in certain soil-growing bacteria such as strains of Rhizobia. This enzyme always consists of two separable proteins called components 1 and 2. Component 1 generally is felt to contain the active site for substrate reduction with 30 Fe and 2 Mo atoms in four complex metal clusters whose structures have recently been determined by Kim and Rees at Caltech using X-ray diffraction techniques. It also has been demonstrated in several laboratories, including our own, that alternative forms of component 1 exist which contain either V and Fe or only Fe instead of Mo and Fe.
One of the goals of our research program is to study the function of these nitrogenase metal clusters in order to gain a better understanding of their roles in the catalytic mechanism of this enzyme. In general, we have used a two-pronged approach to obtain this goal. The first prong is the use of various spectroscopic techniques to probe the electronic, magnetic, and structural environment of the metal clusters. The techniques currently employed are EPR, ENDOR, MCD, Mossbauer, and x-ray absorption spectroscopies.
The second approach used is metal substitution. In general, it is felt that the Mo atoms in component 1 are at or near the active site. To investigate this hypothesis, we have successfully substituted W for Mo in component 1 and shown that this new metal blocks catalysis. We are similarly investigating the function of V and Fe in the alternative forms of the component 1 protein.
R. C. Pollock, H. I. Lee, L. M. Cameron, V. J. DeRose, B. J. Hales. W. H. Orme-Johnson, & B. M. Hoffman, "Detection of CO Bound to Inhibited Forms of Nitrogenase M.-Fe Protein by ENDOR Spectroscopy," J. Amer. Chem. Soc., 1995, 117. 8686-87.
J. Christiansen, R. C. Tittsworth, B. J. Hales, & S. P. Cramer, "Fe and Mo EXAFS of Azotobacter vinelandii Nitrogenase in Partially Oxidized and Singly Reduced Forms," J. Amer. Chem. Soc., 1995, 117, 10017-24.
H-H. T. Nguyen, A. K. Shiemke, S. J. Jacobs, B. J. Hales, M. Lidstrom & S. I. Chan, "The Nature of the Copper Ions in the Membranes Containing the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)," J. Biol. Chem., 1994, 269, 14995-15005.
N. Ravi, V. Moore, S. J. Lloyd, B. J. Hales & B. H. Huynh, "Msssbauer Characterization of the Metal Clusters in Azotobacter vinelandii Nitrogenase VFe Protein," J. Biol. Chem., 1994, 269, 20920-20924.
V. G. Moore, R. C. Tittsworth and B. J. Hales, "Construction and Characterization of Hybrid Component 1 from V-nitrogenase Containing FeMo Cofactor," J. Amer. Chem. Soc., 1994, 116, 12101-12102.