Kinetics and mechanism of superoxide reduction by two-iron superoxide reductase from Desulfovibrio vulgaris

被引:80
作者
Emerson, JP
Coulter, ED
Cabelli, DE
Phillips, RS
Kurtz, DM [1 ]
机构
[1] Univ Georgia, Dept Chem, Athens, GA 30602 USA
[2] Univ Georgia, Ctr Metalloenzyme Studies, Athens, GA 30602 USA
[3] Brookhaven Natl Lab, Dept Chem, Upton, NY 11973 USA
关键词
D O I
10.1021/bi0119159
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Superoxide reductases (SORs) contain a novel square pyramidal ferrous [Fe(NHis)(4)(SCys)] site that rapidly reduces superoxide to hydrogen peroxide. Here we report extensive pulse radiolysis studies on recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborate on our originally proposed scheme for reaction of the [Fe(NHis)(4)(SCys)] site with superoxide [Coulter, E. D., Emerson, J. E., Kurtz, D. M., Jr., and Cabelli, D. E. (2000) J. Am. Chem. Soc. 122, 11555-11556]. This scheme consists of second-order diffusion-controlled formation of an intermediate absorbing at similar to600 nm, formulated as a ferric-(hydro)peroxo species, and its decay to the carboxylate-ligated ferric [Fe(NHis)(4)(SCys)] site with loss of hydrogen peroxide. The second-order rate constant for formation of the 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D2O solvent isotope effect at pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediate does not involve a rate-determining protonation, and are consistent with interaction of the incomina superoxide anion with a positive charge at or near the ferrous [Fe(NHis)(4)(SCys)] site. The rate constant for decay of the 600-nm intermediate follows the pH-dependent rate law: k(2)(obs) = k(2)'[H+] + k(2)" and shows a significant D2O solvent isotope effect at pH 7.7. The values of k(2)' and k(2)" indicate that the 600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order process involving either water or a water-exchangeable proton on the protein at basic pHs. The formation and decay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-type values, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxo ligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participation of the lysyl side chain in directing the superoxide toward the active site and in directing the protonation pathway of the ferric- (hydro)peroxo intermediate toward release of hydrogen peroxide.
引用
收藏
页码:4348 / 4357
页数:10
相关论文
共 32 条
[1]   Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus:: superoxide scavenging by Neelaredoxin [J].
Abreu, IA ;
Saraiva, LM ;
Carita, J ;
Huber, H ;
Stetter, KO ;
Cabelli, D ;
Teixeira, M .
MOLECULAR MICROBIOLOGY, 2000, 38 (02) :322-334
[2]   The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin [J].
Abreu, IA ;
Saraiva, LM ;
Soares, CM ;
Teixeira, M ;
Cabelli, DE .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (42) :38995-39001
[3]   Equilibrium unfolding of dimeric desulfoferrodoxin involves a monomeric intermediate: Iron cofactors dissociate after polypeptide unfolding [J].
Apiyo, D ;
Jones, K ;
Guidry, J ;
Wittung-Stafshede, P .
BIOCHEMISTRY, 2001, 40 (16) :4940-4948
[4]   HIGHLIGHTS OF CURRENT RESEARCH INVOLVING SUPEROXIDE AND PERHYDROXYL RADICALS IN AQUEOUS-SOLUTIONS [J].
BIELSKI, BHJ ;
CABELLI, DE .
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY, 1991, 59 (02) :291-319
[5]   ANALYSIS OF THE TRANSCRIPTIONAL UNIT ENCODING THE GENES FOR RUBREDOXIN (RUB) AND A PUTATIVE RUBREDOXIN OXIDOREDUCTASE (RBO) IN DESULFOVIBRIO-VULGARIS HILDENBOROUGH [J].
BRUMLIK, MJ ;
VOORDOUW, G .
JOURNAL OF BACTERIOLOGY, 1989, 171 (09) :4996-5004
[6]   ISOLATION OF SUPEROXIDE-DISMUTASE MUTANTS IN ESCHERICHIA-COLI - IS SUPEROXIDE-DISMUTASE NECESSARY FOR AEROBIC LIFE [J].
CARLIOZ, A ;
TOUATI, D .
EMBO JOURNAL, 1986, 5 (03) :623-630
[7]  
CLAY MD, 2001, J AM CHEM SOC
[8]  
COEHLO AV, 1997, J BIOL INORG CHEM, V2, P680
[9]   NADH peroxidase activity of rubrerythrin [J].
Coulter, ED ;
Shenvi, NV ;
Kurtz, DM .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1999, 255 (02) :317-323
[10]   A role for rubredoxin in oxidative stress protection in Desulfovibrio vulgaris:: Catalytic electron transfer to rubrerythrin and two-iron superoxide reductase [J].
Coulter, ED ;
Kurtz, DM .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 2001, 394 (01) :76-86