9305399 Stolz The strictly anaerobic microbe, Geobacter metallireducens (GS-15) is a dissimilatory iron reducing bacterium which couples the oxidation of short chain fatty acids and aromatic compounds to the reduction of Fe(III), nitrate, Mn(IV), or U(VI). This type of organism is extremely important in the oxidation of organics and the reduction of inorganics and metals in anaerobic soil, aquifers, and estuarine sediments. Nitrate induces both nitrate reductase (NR) and the overexpression of five c-type cytochromes. This NR is stable under oxic conditions and its inhibition by oxygen is reversible. It appears to contain three polypeptides of Mr 18, 36, and 43 kDa and three c-type cytochromes (Mr 28, 46, and 68 kDa), and is not related to the NRs from other nitrate-respiring or denitrifying bacteria. The objective of this proposal is to fully characterize this novel NR and determine which cytochromes are involved in nitrate reduction. The NR and cytochromes will be purified, native structure, midpoint potentials, substrate specificity, and kinetics will be determined, and antibodies will be raised against each purified macromolecule. The pathway of nitrate reduction and electron transport will be deduced from the data. Future studies will use the antibodies as a taxonomic tool in comparative studies and as environmental probes. %%% Geobacter metallireducens is a bacterium which grows by coupling the oxidation of organic carbon to the reduction of iron in the total absence of oxygen. The type of organism is extremely important in anoxic soils, aquifers, and estuarine sediments, since it also metabolizes organic pollutants like benzene and toluene, and inorganic contaminants, such as nitrate, manganese, and uranium. Preliminary work has shown that the enzyme that converts nitrate to nitrite in this cell is novel and its function involves c-type cytochromes. The purpose of this work is then to characterize the novel nitrate reductase and then determine th e cytochrome(s) involved in nitrate reduction. The results from this study will help elucidate this unusual process of nitrate reduction, and provide the background for future studies on other unique biochemical pathways in this remarkable organism. ***
