PROJECT SUMMARY/ABSTRACT Pseudomonas aeruginosa (PA) is a ubiquitous Gram-negative bacteria and opportunistic pathogen that is of considerable medical importance due to its multidrug resistance and tendency to form antibiotic resistant biofilms. During PA infection, a strong innate immune response is generated by phagocytes, including neutrophils and macrophages, in the form of the respiratory burst (RB). Although many bactericidal agents are generated during the RB, a leading DNA-damaging and PMF (proton motive force) perturbing oxidant is hydrogen peroxide (H2O2). PA is exposed to phagocyte-derived H2O2 in a number of disease settings, including cystic fibrosis, chronic obstructive pulmonary disease, burns/blast/surgical/diabetic wounds and urinary tract infections. PA defense against H2O2 is governed by the DNA-binding transactivator OxyR. Of the numerous PA genes under OxyR control, optimal resistance to H2O2 requires activation of a two gene operon encoding katb and ankb, which encode two proteins strategically deployed to the periplasm. KatB is a group 1 heme b-type catalase that converts H2O2 into H2O and O2 and AnkB is a putative ankyrin repeat protein of unknown function. The overall goal of this multi-PI exploratory R21 application is to combine the structural, biophysical, and biochemical expertise of the Kovall lab with the microbial physiology, biofilm, and genetics expertise of the Hassett lab in order to elucidate the novel functions of AnkB and KatB in response to exogenous H2O2. Our preliminary data supports the hypothesis that AnkB is a novel heme binding protein that is required to transfer heme to monomeric KatB in order to facilitate the formation of catalytically active KatB tetramers. This mechanism is likely an important feature of the in vivo confrontation between PA and the H2O2 component of the RB mediated by phagocytic cells during PA infection. To achieve our overall goal and test our hypothesis, we will pursue the following two specific aims. Aim 1: Determine the structures and define the functions of AnkB and KatB. Aim2: Determine the role of AnkB and KatB in planktonic vs. biofilm-forming PA in response to H2O2. Completion of these exploratory, structural, mechanistic, and highly translational studies will begin to uncover the role of AnkB and KatB in protecting PA from H2O2 and will likely lead to important functional insights into the overall behavior of PA during infection associated with various important human diseases.