Mississippi State University
Date of Degree
Graduate Thesis - Open Access
College of Arts and Sciences
Department of Biological Sciences
The enzyme nitrogenase catalyzes the energy-dependent reduction of dinitrogen to ammonia via biological nitrogen fixation. Nitrogenase is composed of two metalloproteins known as the molybdenum-iron (MoFe) protein and the iron (Fe) protein. The Fe protein, a 60-kDa dimer of the product of the nifH gene, contains a single 4Fe-4S cluster and two Mg-ATP-binding sites, one at each subunit. The Fe protein is the obligate electron donor to the MoFe protein. To date, no other mutual protein has shown to substitute Fe protein in biological fixation, and the NifH is functional only in the presence of the nifessory protein NifM. Interestingly, the protochlorophyllide reductase (ChlL) encoded by the chlL gene of Chlamydomonas reinhardtii shows significant homology and structural similarity with NifH. Previously, our laboratory has shown that the ChlL can substitute the Fe protein in the functioning nitrogenase only in the absence of NifM. We have also shown that the NifM is a PPIase and the Pro-258 located in the C-terminus of NifH is one of the substrates for NifM. Since the least structural homology exists between NifH and ChlL at the C-terminal region, we hypothesized that we can generate a NifM-independent NifH-ChlL chimeric protein by replacing the C-terminus of NifH (that spans the substrate of PPIase) with that of ChlL. To test this idea we created a chimeric construct by replacing the NifH C-terminal region (residues 248-291) with the ChlL C-terminal region (residues 240-294). The chimeric gene was then transformed into the nifM- Azotobacter vinelandii strain AV98. While the wild type nifH could not render a Nif+ phenotype to the nifM- AV98, the chimera could impart Nif+ phenotype to this nifM- strain. This result demonstrated that the NifH-ChlL chimeric protein is NifM-independent.
Harris, Kelvin, "Understanding the NifM Dependence of NifH in Azotobacter Vinelandii: Functional Substitution of NifH by a NifH-ChlL Chimeric Construct in a NifM- Strain" (2007). Theses and Dissertations. 4844.