Superoxide reductase from Desulfoarculus baarsii: reaction mechanism and role of glutamate 47 and lysine 48 in catalysis.
Superoxide reductase (SOR) is a small metalloenzyme that catalyzes reduction of O(2)(*)(-) to H(2)O(2) and thus provides an antioxidant mechanism against superoxide radicals. Its active site contains an unusual mononuclear ferrous center, which is very efficient during electron transfer to O(2)(*)(-) [Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121]. The reaction of the enzyme from Desulfoarculus baarsii with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bimolecular reaction of superoxide reductase with superoxide, with a rate constant of (1.1 +/- 0.3) x 10(9) M(-1) s(-1). A first intermediate is formed which is converted to a second one at a much slower rate constant of 500 +/- 50 s(-1). Decay of the second intermediate occurs with a rate constant of 25 +/- 5 s(-1). These intermediates are suggested to be iron-superoxide and iron-peroxide species. Furthermore, the role of glutamate 47 and lysine 48, which are the closest charged residues to the vacant sixth iron coordination site, has been investigated by site-directed mutagenesis. Mutation of glutamate 47 into alanine has no effect on the rates of the reaction. On the contrary, mutation of lysine 48 into an isoleucine led to a 20-30-fold decrease of the rate constant of the bimolecular reaction, suggesting that lysine 48 plays an important role during guiding and binding of superoxide to the iron center II. In addition, we report that expression of the lysine 48 sor mutant gene hardly restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions.
Lombard
M
M
Laboratoire de Chimie et Biochimie des Centres Redox Biologiques, DBMS-CEA/CNRS/Université Joseph Fourier, 38054 Grenoble, Cedex 9, France.
Houée-Levin
C
C
Touati
D
D
Fontecave
M
M
Nivière
V
V
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002384:Catalysis; D002417:Cattle; D003901:Desulfovibrio / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011675:Pulse Radiolysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000235:genetics; D013481:Superoxides / Q000378:metabolism*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007532:Isoleucine; D013481:Superoxides; D018698:Glutamic Acid; D010088:Oxidoreductases; C121849:superoxide reductase; C095500:SodA protein, Bacteria; C087504:SodB protein, Bacteria; D013482:Superoxide Dismutase; D008239:Lysine