Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase

Abstract

Rubrerythrin was purified by multistep chromatography under anaerobic, reducing conditions from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimer with a molecular mass of 39.2 kDa and contains 2.9 ؎ 0.2 iron atoms per subunit. The purified protein had peroxidase activity at 85°C using hydrogen peroxide with reduced P. furiosus rubredoxin as the electron donor. The specific activity was 36 mol of rubredoxin oxidized/min/mg with apparent K m values of 35 and 70 M for hydrogen peroxide and rubredoxin, respectively. When rubrerythrin was combined with rubredoxin and P. furiosus NADH:rubredoxin oxidoreductase, the complete system used NADH as the electron donor to reduce hydrogen peroxide with a specific activity of 7.0 mol of H 2 O 2 reduced/min/mg of rubrerythrin at 85°C. Strangely, as-purified (reduced) rubrerythrin precipitated when oxidized by either hydrogen peroxide, air, or ferricyanide. The gene (PF1283) encoding rubrerythrin was expressed in Escherichia coli grown in medium with various metal contents. The purified recombinant proteins each contained approximately three metal atoms/subunit, ranging from 0.4 Fe plus 2.2 Zn to 1.9 Fe plus 1.2 Zn, where the metal content of the protein depended on the metal content of the E. coli growth medium. The peroxidase activities of the recombinant forms were proportional to the iron content. P. furiosus rubrerythrin is the first to be characterized from a hyperthermophile or from an archaeon, and the results are the first demonstration that this protein functions in an NADH-dependent, hydrogen peroxide: rubredoxin oxidoreductase system. Rubrerythrin is proposed to play a role in the recently defined anaerobic detoxification pathway for reactive oxygen species.

Distinguishing features of δ-proteobacterial genomes

“…Of the 46 RP genes Ն80 aa length of the BDEBA genome, 45 are PHX, a high proportion consistent with the proposition that BDEBA is fast growing (17). DESVU contains several PHX anaerobic detoxification genes, including two rubrerythrin genes and two rubredoxin oxidoreductase genes that can protect the organism against oxidative stress or other reactive toxins (18,19). The gene of highest PHX level [E(g) ϭ 1.90] is the large ribosomal protein gene S1.…”

“…Rubrerythrin (Rbr), with a di-iron active site, is found often PHX in anaerobic and microaerophilic bacteria and in archaeal organisms. The presence of Rbr is interpreted as an oxidative stress protection system in air-sensitive bacteria and archaea (18,19). DESVU and DESPS are obligate anaerobic bacteria but appear to possess oxygen-reducing systems.…”

“…3 and 5; see also supporting information in Tables 12 and 15 with ␦-genome lists of PHX genes). Rubrerythrin (Rbr), Nigerythrin, and Rubredoxin oxidoreductase were recently described as alternative oxidation stress protection proteins functioning in an anaerobic environment (18,19). Rbr is widespread in archaea and some bacteria, particularly in organisms that die in the presence of oxygen.…”

“…Rbr is widespread in archaea and some bacteria, particularly in organisms that die in the presence of oxygen. The function of Rbr has been debated, and recent evidence both in vivo and in vitro strongly supports Rbr in a role of a novel oxidative stress protection system (18,19). A strictly anaerobic organism needs to eliminate oxygen radicals that arise during reduction of SO 4 A to SO Ϫ .…”

“…Genomic analyses have shown that Rbr-like proteins are ubiquitous among archaea and bacteria and in genomes of many anaerobes that encode multiple Rbr homologues. The main function of Rbr seems to be reduction of hydrogen peroxide (19). TonB-dependent receptors.…”