Abstract
Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens. It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein reduces superoxide very efficiently (k = 1.5 x 10(9) m(-1)s(-1)), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted approximately 50 nm from the absorption of the resting ferric state. The role 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.
Highlights
Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens
We present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis
This protein reduces superoxide very efficiently (k ؍1.5 ؋ 109 M؊1s؊1), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes
Summary
Construction of Escherichia coli Transformants for the Expression of Neelaredoxin Mutants—A pT7-7 plasmid containing the Nlr gene (pT7AfNlr) [9] was used as a template in a site-directed mutagenesis assay to create an E12V and an E12Q mutation in Nlr (plasmids pT7AfNlrE12V and pT7AfNlrE12Q), using the QuikChangeTM Sitedirected Mutagenesis kit from Stratagene. All kinetic data obtained are consistently proportional to the iron content of the samples, showing that there is no effect due to the presence of zinc. Redox titrations were performed under aerobic conditions and monitored by visible spectroscopy (400 – 820 nm), using a protein concentration sufficient to have a 660 nm band with at least 0.2 of absorbance. Nlr has a tendency to become re-reduced under anaerobic conditions, leading to redox titrations with a bad equilibrium. The solution of Nlr was prepared in a N2O atmosphere and in the presence of formate as 1⁄7OH scavenger, as described in Reactions 5. The final models of the oxidized and reduced states had 90% and 89% of the
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