Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus:: superoxide scavenging by Neelaredoxin

Archaeoglobus fulgidus is a hyperthermophilic sulphate-reducing archaeon. It has an optimum growth temperature of 83 degreesC and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O-2 .(-)) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O-2 .(-) scavenger in A. fulgidus, by studying both the wild-type and recombinant proteins. Nlr is a 125-amino-acid blue-coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium-stained gel assays of cell-soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O-2 .(-). Furthermore, it is shown that Nlr is able to both reduce and dismutate O-2 .(-), thus having a bifunctional reactivity towards O-2 .(-). Kinetic and spectroscopic studies indicate that Nlr's superoxide reductase activity may allow the cell to eliminate O-2 .(-) quickly in a NAD(P)H-dependent pathway. On the other hand, Nlr's superoxide dismutation activity will allow the cell to detoxify O-2 .(-) independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg(-1) by the xanthine/xanthine oxidase assay at 25 degreesC. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83 degreesC, the rate constant is 5 x 10(6) M-1 s(-1). Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.