The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin

Neelaredoxin is a mononuclear iron protein widespread among prokaryotic ana erobes and facultative aerobes, including human pathogens. It has superoxid e scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reacti on of superoxide with the reduced form of neelaredoxin from the hyperthermo philic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein re duces superoxide very efficiently (k = 1.5 x 10(9) M-1 s(-1)), and the dism utation activity is rate-limited, in steady-state conditions, by the much s lower superoxide oxidation step. These data show unambiguously that the sup erfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presen ts a novel type of reactivity toward superoxide, a result of particular rel evance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced b y the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on t he catalytic mechanism was obtained by the detection of a transient interme diate ferric species upon oxidation of neelaredoxin by superoxide, detectab le by visible spectroscopy with an absorption maximum at 610 nm, blueshifte d similar to 50 nm from the absorption of the resting ferric state. The rol e of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.