Interplay of conformational and redox properties of cytochromes in disease

About This Grant

This project focuses on characterization of heme enzymes that function in electron-rich environments and proteins they interact with. Bacteria encounter low or no O2 in a variety of habitats and such environments are particularly common at sites of infection and in biofilms. At low O2, microbes have evolved to keep using this powerful electron acceptor to benefit their energy production. At no O2, nitrite is frequently employed and there are multiple pathways to process this electron acceptor, which optimizes electron flux. Heme enzymes having multiple redox sites are critical players in these multielectron transformations. Recent studies as well as analyses of rapidly rising genomic sequences suggest elaborate regulation of electron flow in these enzymes and features of the active sites that have not yet been characterized. Understanding intra- and interprotein regulation mechanisms and deciphering molecular details that define reactivity in bacterial redox enzymes are critical for guiding the design of new antibiotic therapies. Our recent efforts have focused on characterization of conformational dynamics and redox reactivity of cytochrome enzymes and proteins through interactions with membranes and on regulatory mechanisms to deal with the limited supply of O2 under microoxic conditions, to coordinate electron flow and substrate delivery. We have identified multiple metalloproteins critical for the microoxic function of cbb3 oxidases in Pseudomonas bacteria, elucidated interactions and redox cooperativity of their electron donors, and characterized the ability of these enzymes to reduce NO. We have initiated studies of nitrite reduction in a previously uncharacterized clade of NrfA enzymes and their mimics and also identified protein systems to enable spectroscopic studies of reaction intermediates during NO reduction. In the next five years, we will (1) develop understanding of the redox function of Pseudomonas cytochrome c5 and its regulation; (2) characterize catalytic activity and electron flow in Campylobacter jejuni and Helicobacter pullorum NrfA to establish reactivity differences between Lys- and His-ligated NrfA active sites; and (3) characterize enzymatic activity and reaction intermediates of heme enzymes that reduce NO. To probe the interplay of conformational dynamics and redox function in these systems, we will combine in vitro structural, spectroscopic, and electrochemistry studies with analyses of protein interactions and bacterial phenotypes in vivo. This multidisciplinary program will yield fundamental insights about mechanisms of redox enzymes relevant to bacterial fitness and pathogenesis.