9727982 Campbell Biochemistry of eukaryotic nitrate reductase (NR; EC 1.6.6.1-3), catalyzing NADH/NADPH driven reduction of nitrate to nitrite in plants, algae and fungi, has been studied in this laboratory for 20 years. The investigator has studied structure and function of NR, including modeling part of its three-dimensional structure. Natural sources provide only small amounts of NR and to advance biochemical understanding, recombinant expression of NR and active fragments has been successfully used. The investigation will involve recombinant expression of active holo-NR in Pichia pastoris, a methylotrophic yeast, for producing purified enzyme needed to obtain complete 3-D structure. Mutant forms of NR will be used to study functionality in the Mo-molybdenum pterin cofactor binding site (Mo-MPT), nitrate reducing active site and subunit interface. A model of NR is being derived using the sulfite oxidase 3-D structure. Studies of NR's catalytic mechanism via spectral and steady-state kinetic analyses will be done with pre-steady state kinetics and EPR spectra done via collaboration. Existing mutants of ligands to Mo-MPT will be analyzed in comparison to wild-type enzyme via x-ray absorption spectroscopy at the Stanford Synchrotron Radiation Laboratory. These studies will yield a complete and detailed picture of NR structure and function and guide future studies. Nitrate reductase is a unique member of a multicenter redox enzyme family; these catalyze long-distance electron transfer. NR is important in agriculture since it serves as the first enzymatic step in nitrogen accumulation by plants. NR also has importance in biotechnology for addressing environmental problems arising from nitrate pollution of ground and surface waters. NR is a "green" catalyst for nitrate test kits for analysis of water and part of a system for eliminating nitrate pollution of potable water via enzymatic denitrification. Basic research on NR is paying off for society since it is a tool in environmental biotechnology.
