7 - Kinetic Characterization of the Redox Regulation of OhrR

Abstract

Pseudomonas aeruginosa is a pathogen that needs to cope with the oxidative insult imposed by the mammalian host. Organic Hydroperoxide Resistance protein (Ohr) is a Cys-based peroxidase that together with its transcriptional regulator Organic Hydroperoxide Resistance Regulator (OhrR) comprises a system that is central to the P. aeruginosa response towards organic hydroperoxides and peroxynitrite. Expression of Ohr gene is regulated by OhrR, which has the ability to bind to DNA, modulated by the oxidation of a critical Cys residue. Therefore, we investigate the kinetics of OhrR oxidation and reduction by the changes of its intrinsic fluorescence. The second-order rate constant for the reactions of OhrR from P. aeruginosa (PaOhrR) with tert-butyl hydroperoxide (tBHP) and with fatty acid hydroperoxides were in the 103 M-1 s-1 and in the 106M-1 s-1ranges, respectively. The second-order rate constant for the reaction between OhrR and peroxynitrite was determined as 6x105 M-1 s-1by initial rate approach. No redox dependent change in the fluorescence of PaOhrRC19S was observed, which is consistent with the notion that Cys19 is the reactive Cys. The OhrR from Chromobacterium violaceum (CvOhrR) was also investigated as its redox dependent ability to bind DNA was previously characterized. As PaOhrR, CvOhrR also reacted with tBHP with a rate constant in the 103 M-1 s-1 range. In contrast, reaction of CvOhrR with oleic acid hydroperoxide was considerably slower (103 M-1 s-1) than the equivalent reaction of PaOhrR. Concerning the reduction of CvOhrR, DTT and lipoamide were very poor reductants, whereas thioredoxin A (TrxA) reacted with the transcription factor with rate constants in the 104 M-1 s-1 range. Currently, we are investigating whether the binding of CvOhrR to its target DNA might affect its oxidation by distinct hydroperoxides. The kinetic characterization of OhrR proteins can improve our knowledge on the mechanisms underlying the response of bacteria to fatty acid hydroperoxides and peroxynitrite, two oxidants present at the host pathogen interface.