The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin.
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.
Abreu
I A
IA
Instituto de Tecnologia Quimica e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, APT 127, 2780-156 Oeiras, Portugal.
Saraiva
L M
LM
Soares
C M
CM
Teixeira
M
M
Cabelli
D E
DE
D019611:Archaeoglobus fulgidus / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D033862:Iron-Binding Proteins; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013696:Temperature; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D002352:Carrier Proteins; D033862:Iron-Binding Proteins; D008024:Ligands; D011994:Recombinant Proteins; D013481:Superoxides; D018698:Glutamic Acid; D006861:Hydrogen Peroxide; D007501:Iron; D010088:Oxidoreductases; D013482:Superoxide Dismutase; C414179:nlr protein, Desulfovibrio gigas