FLAVIN-DEPENDENT ALKYL HYDROPEROXIDE REDUCTASE FROM SALMONELLA-TYPHIMURIUM .1. PURIFICATION AND ENZYMATIC-ACTIVITIES OF OVEREXPRESSED AHPF AND AHPC PROTEINS

The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and pur ified from Escherichia coli For investigations of their catalytic prop erties. Recombinant proteins were isolated in high yield (25-33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E. col i cells. We have developed quantitative enzymatic assays for AhpF alon e and for the combined AhpF/AhpC system which have allowed us to addre ss such issues as substrate specificity and inhibition by thiol reagen ts for each protein, All assays gave identical results whether overexp ressed S. typhimurium proteins from E. coli or proteins isolated direc tly from S. typhimurium were used. Anaerobic hydroperoxide reductase a ssays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide , and hydrogen peroxide can all be reduced by the combined enzyme syst em. AhpF possesses multiple pyridine nucleotide-dependent activities [ 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase, oxidase, transhy drogenase, and, in the presence of AhpC, peroxide reductase activities ]. Although AhpF can use either NADH or NADPH as the electron donor fo r these activities, NADH is the preferred reductant (K-m,K-app of AhpF for NADH was more than 2 orders of magnitude lower than that for NADP H when analyzed using DTNB reductase assays). Thiol-modifying reagents react readily with each reduced protein, leading to complete loss of hydroperoxide and DTNB reductase activities. In contrast, thiol modifi cation of reduced AhpF does not affect transhydrogenase or oxidase act ivities. These data provide the first direct evidence for a catalytic mechanism for peroxide reduction involving redox-active disulfides wit hin each protein.