Abstract

Bacteria have developed complex strategies to detoxify and repair damage caused by reactive oxygen species. These compounds, produced during bacterial aerobic respiration as well as by the host immune system cells as a defense mechanism against the pathogenic microorganisms, have the ability to damage nucleic acids, proteins and phospholipid membranes. Here we describe the crystal structure of Pseudomonas aeruginosa Ohr, a member of a recently discovered family of organic hydroperoxide resistance proteins. Ohr is a tightly folded homodimer, with a novel / fold, and contains two active sites located at the monomer interface on opposite sides of the molecule. Using in vitro assays, we demonstrate that Ohr functions directly as a hydroperoxide reductase, converting both inorganic and organic hydroperoxides to less toxic metabolites. Site-directed mutagenesis confirms that the two conserved cysteines in each active site are essential for catalytic activity. We propose that the Ohr catalytic mechanism is similar to that of the structurally unrelated peroxiredoxins, directly utilizing highly reactive cysteine thiol groups to elicit hydroperoxide reduction.