9630127 Dean Iron sulfur (Fe-S) clusters are found in numerous proteins that have important redox, catalytic or regulatory properties. Such clusters are known to act as electron carriers and environmental sensors, or to be involved in substrate binding and catalysis, yet very little is known about how they are formed biologically. Because free intracellular Fe and S are extremely toxic and are unlikely to exist in the cell in appreciable quantities to permit spontaneous Fe-S cluster formation, it is likely that biological Fe-S cluster formation occurs through a controlled process catalyzed by a specific set of enzymes. Three enzymes associated with nitrogen fixation (NifS, NifU and ORF6) are likely to have functions related to the mobilization and activation of Fe and S for Fe-S cluster formation. Recent sequence comparisons have shown that these enzymes are among the most conserved throughout nature. This project will analyze the function of these enzymes by determining how they work together to form biologically active Fe-S clusters. This goal will be accomplished by determining the interaction among these proteins and by optimizing conditions which leads to their ability to catalyze Fe-S clusters in vitro. %%% Iron(Fe) and sulfur(S) are essential elements for all free-living organisms. In some cases, Fe and S are brought together to form structures called Iron-Sulfur clusters. These clusters are important biological entities because they are involved, for example, as electron carriers in important life sustaining processes such as photosynthesis, nitrogen fixation, and respiration. Because iron and sulfur are extremely toxic, the cell must be able to form Fe-S clusters through a controlled enzymatic process. Three enzymes have been identified that are likely involved in the biological formation of Iron-Sulfur clusters. In this project, purified samples of these enzymes will be used to determine how they work together to form such clusters. *** Project Summary Iron-Sulf ur clusters (Fe-S clusters) are found in numerous proteins that have important redox, catalytic or regulatory properties. Fe-S clusters are known to act as electron carriers, environmental sensors, or to be involved in substrate binding and catalysis. However, very little is known about how they are formed biologically. Because free Fe and S are toxic and are unlikely to exist in the cell in appreciable quantities, it is likely that biological Fe-S cluster formation occurs through a controlled process catalyzed by a specific set of enzymes. Previous work using the nitrogenase system has indicated that three nif-specific gene products from the nitrogen-fixing organism Azotobacter vinelandii are good candidates to participate in the formation of the Fe-S cores required for nitrogenase activation. These genes are nif~, nifU, and a gene designated orf~. NifS has been shown to be a cysteine desulfurase which is able to catalyze Fe-S cluster formation in vitro, whereas the specific functions of NifU and Orf6 have not yet been established. However, the phenotype of nifU mutants and the organization of conserved cysteine residues contained within NifU and Orf6 indicate they could have roles in the activation of Fe for Fe-S cluster assembly, act as intermediate Fe/S carriers, or provide a scaffolding site for Fe-S cluster formation. The goals of the present project are to determine how gi~o~ge~ F~S clusters are formed biologically and to find out whether or not the'~'mo'lecular mechanism for nit'rogenase~Fe-S cluster fo~madon represents a global mechanism for formation of Fe-S clusters found in other proteins. The reason for suspecting that NifS, NifU and Orf6 activities might represent general mec~anisms for Fe-S cluster formation is that homologs to all three proteins have been identified in other prokaryotic and eucaryotic organisms that do not fix pitrogen. The potential Ioles of NW, NifS and Orf6 in nitrogenase specific Fe-S cluster formation will be explored by the following experiments. First, co nditions for p~rotein-protein interactions between and among NifU, NifS, Orf6, and an apo-form of the mtrogenase Fe protein will be examined. This will be accomplished primarily by chemical~DI~ eLues and affinity chromatography using the purified proteins. Second, the potential interaction among NifU, NifS, Orf6, and the apo-Fe protein with other proteins whose identity is not yet known will be determined. This will be done using affinity chromatography and nitrogenase-derepressed crude extracts. Third, conditions for the optimum in vitro catalytic formation of Fe-S clusters will be determined using various combinations of purified NifU, NifS, orf6 and an apo-form of the nitrogenase Fe protein. Fe-S cluster assembly will be monitored by activation of nitrogenase Fe protein activity and by various spectroscopic techniques including Electron Paramagnetic Resonance, Resonance Raman, Magnetic Circular Dichroism, and ~s'sb'auer spectroscopies. Fourth, crude extracts will be used to determine if proteins other than NifU, NifS, and Orf6 might participate in nitrogenase Fe-S cluster formation. Fifth, the possible formation of Fe-S cluster intermediates, both in the presence and absence of the apo-form of the nitrogenase Fe protein, will be monitored by a series of time-course freeze-quench experiments where assembly is monitored by various spectroscopic techniques. The second major goal of the project is to determine if homologs to NifU, NifS, and Orf6 are contained within the A. vinelandii genome and, if so, do they encode housekeeping functions for general Fe-S cluster assembly. A nifS homolog has already been identified. In the present project the chromosomal region encoding the NifS-like protein, which is suspected to also include nipJ-like and orl~like genes, will be cloned and characterized. The functions of these genes will be tested using gene directed mutagenesis techniques. If, as is expected, one or more of these genes is essential for viability, genetic constructions will be perfo~med to permit their conditional expression. 3
