Pentaheme cytochrome c nitrite reductase (NrfA) catalyzes the remarkable six-electron reduction of NO2− to NH4+ in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway. Our work focuses on NrfA and its membrane-bound redox partner NrfH from Geobacter lovleyi, a DNRA bacterium identified for its environmental relevance. Consistent with all previous structurally characterized NrfAs, we found that G. lovleyi NrfA crystallizes as a homodimer with each subunit containing five c-type hemes. Four of the hemes are bis-His coordinated and function as electron storage and/or transfer units. The fifth heme, which constitutes the active site, is only 5-coordinate and is bound by Lys as the axial ligand. This unique active site lysine is hypothesized to create a more positive reduction potential at the active site for rapid electron transfer and/or to aid in the binding of anionic substrates.
A primary objective of this project is to examine the activity and electron flow of the multiheme NrfA-NrfH complex. We are currently focused on understanding communication between NrfA monomers via Heme 5, which sits at the dimer interface, and the role of NrfH as intermediary between NrfA and the quinol pool. A secondary objective is to study NrfA inside of bacterial microcompartments (BMCs), protein-based organelles that encapsulate and organize enzymes and other proteins. We aim to understand the impact of confinement on catalysis for redox enzymes and to generate a biofactory within this microenvironment.
To accomplish these objectives, we are employing a synergistic combination of biochemical, kinetic, spectroscopic, and electrochemical methods to examine how NrfA stores and regulates the flow of electrons. Ultimately, our work will lay the foundation for subsequent detailed mechanistic studies to ascertain how this unique pentaheme enzyme orchestrates the multi-electron and multi-proton reduction of NO2− to NH4+.