Ammonium is an important nitrogen source for plants and microbial communities and is a main component in nitrogen-rich fertilizer. Unfortunately, ammonium can be readily converted to nitrite and nitrate which leach more easily from soils than ammonium. Certain organisms, however, can metabolically utilize nitrate and nitrite and convert them back to ammonium via a pathway known as dissimilatory nitrate reduction to ammonium (DNRA). One of the protein complexes involved in the DNRA pathway is composed of two proteins: NrfH, a membrane-bound quinol oxidase, and its redox partner NrfA, a soluble periplasmic protein that catalyzes the six-electron, eight-proton reduction reaction. This pathway appears to help retain nitrogenous nutrients in the soil, but the mechanistic details involved in this biotic chemical conversion remain largely unknown. Theoretical calculations over the past several years have proved invaluable for predicting the reaction intermediates and helping inform biochemical efforts to unravel the reaction pathway. Our lab has successfully overexpressed and characterized NrfA from Geobacter lovleyi and obtained a crystal structure of this protein. To study the mechanism of nitrite reduction at the NrfA active site in the presence of its physiologically relevant electron donor, we must also overexpress NrfH. NrfH is a membrane bound protein with a soluble, globular head which contains four c-type, bis-His hemes. NrfH receives electrons from the membrane quinol pool and transfers them to NrfA for catalysis. Currently, there is only one crystal structure of NrfH, and this protein has never been successfully overexpressed. Therefore, our lab has created several methods for overexpressing and isolating NrfH. Once NrfH is successfully overexpressed, we will analyze the electron movement from a physiologically relevant redox partner to NrfA, enabling us to better characterize the loading of reducing equivalents to the active site in a more native environment.

Overexpressing NrfA and its redox partners for mechanistic studies

Fall 2022 abstracts, posters, and presentations will launch in mid-September.

ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.