Rational improvement of the activity of a nitrite reductase model

University of Minnesota M.S. thesis. May 2014. Major: Chemistry. Advisor: Steven Berry. 1 computer file (PDF); v, 74 pages.Nitrite reductase (NiR) is a bacterial enzyme that catalyzes the one electron reduction of nitrite (NO2-) to nitric oxide (NO). Through site-directed PCR mutagenesis, variants of the electron transfer protein azurin (See figure) were rationally designed to mimic Nitrite Reductase (NiR) in an attempt to increase our knowledge about enzyme function and design. Several variants were created by incorporation of a Type 2 copper center on the surface of the protein. Further mutations were added to aid in electron transfer as well as to match the potential of the Type 1 copper in azurin to that of the Type 1 copper in NiR. Each variant was characterized using EPR, UV-Vis and mass spectroscopy. Finally, we characterized each variant using Michaelis-Menten kinetics. This activity was determined using the Griess assay, which allows us to spectrophotometrically quantify the amount of nitrite in our reaction. We compared the activity of our NiR models. The results show that the variant designed for faster electron transfer did not increase the activity of our enzyme, but did increase activity when added with the other mutations. The two variants designed to decrease the reduction potential of the Type 1 copper site were found to increase the activity by themselves, but decrease the activity when combined