Functional Evaluation of the Mechanisms of Reactive Oxygen Detoxification in the Hyperthermophilic Archaeon Pyrococcus Furiosus Using Complementation Studies

Abstract

Pyrococcus furiosus is an obligatory anaerobic hyperthermophilic archaeon (optimal growth temperature is 100&deg;C ) which can be isolated from deep and shallow marine hydrothermal vent systems. Initially, due to the obligatory anaerobic nature of P. furiosus, it was thought that this organism did not possess the ability to detoxify reactive oxygen species. Recent studies, however, have shown that P. furiosus produces the enzyme, superoxide reductase (SOR) which catalyzes the reduction of the reactive oxygen specie, superoxide, to hydrogen peroxide. This is in contrast to aerobic organisms which use superoxide dismutase (SOD) to convert superoxide to hydrogen peroxide and O2. Surprisingly, P. furiosus SOR, unlike many P. furiosus enzymes, was shown to function at low temperature (<25o C). A model for superoxide reduction by SOR has been developed by Jenney et al. (1999) where the electrons used by SOR to reduce superoxide are supplied by the Fe-S protein, rubredoxin (Rd) and Rd is reduced by the oxidoreductase, NAD(P)H-rubredoxin oxidoreductase (NROR). Previous studies have shown that SORs from Desulfovibrio vulgaris and Desulfovibrio gigas can complement defects in Escherichia coli superoxide dismutases (sodA, sodB). In addition, E. coli has a gene, norW that is homologous to P. furiosus NROR (42% similarity) and also has the gene, norV which codes for a protein containing a rubredoxin domain that is 49% similar to P. furiosus Rd. The norV and norW genes exist together as part of an operon. In this study, P. furiosus SOR, Rd, NROR genes which are thought to be involved in SOR detoxification of O2- were cloned and expressed in E. coli strain JM105. Using complementation studies, P. furiosus SOR was shown to detoxify O2- generated in the E. coli sodA, sodB strain (NC906) and restore growth under formerly nonpermissive conditions. E. coli strains with deletion of the putative flavorubredoxin (norV) and NROR (norW) genes have been constructed and verified by PCR. P. furiosus SOR has shown a low level of complementation in the E. coli sodA, sodB, and norV mutant suggesting that the E. coli flavorubredoxin (Fl-Rd) may be supplying the electrons to recombinant P. furiosus SOR in vivo.