Desulfovibrio alcoholovorans sp. nov., a sulfate-reducing bacterium able to grow on glycerol, 1,2- and 1,3-propanediol

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Superoxide reductase as a unique defense system against superoxide stress in the microaerophile Treponema pallidum.

International audienceAerobic life requires the presence of antioxidant enzymes, such as superoxide dismutase, catalase, and peroxidase to eliminate deleterious oxygen derivatives. Treponema pallidum, a microaerophilic bacterium responsible for venereal syphilis, is an interesting organism because it lacks all of the above-mentioned enzymes, as deduced from its recently sequenced genome. In this paper, we describe a gene in T. pallidum with sequence homologies to a new class of antioxidant systems, named superoxide reductases, recently isolated from sulfate-reducing bacteria (Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121). We report that (i) expression of the T. pallidum gene fully restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions; (ii) the corresponding protein displays a strong superoxide reductase activity; and (iii) the T. pallidum protein contains only one mononuclear nonheme ferrous center, able to reduce superoxide selectively and efficiently, whereas previously characterized superoxide reductase from Desulfoarculus baarsii contains an additional rubredoxin-like ferric center. These results suggest that T. pallidum antioxidant defenses rely on a new class of superoxide reductase and raise the question of the importance of superoxide reductases in mechanisms for detoxifying superoxide radicals

Reaction of the desulfoferrodoxin from Desulfoarculus baarsii with superoxide anion. Evidence for a superoxide reductase activity.

International audienceDesulfoferrodoxin is a small protein found in sulfate-reducing bacteria that contains two independent mononuclear iron centers, one ferric and one ferrous. Expression of desulfoferrodoxin from Desulfoarculus baarsii has been reported to functionally complement a superoxide dismutase deficient Escherichia coli strain. To elucidate by which mechanism desulfoferrodoxin could substitute for superoxide dismutase in E. coli, we have purified the recombinant protein and studied its reactivity toward O-(2). Desulfoferrodoxin exhibited only a weak superoxide dismutase activity (20 units mg(-1)) that could hardly account for its antioxidant properties. UV-visible and electron paramagnetic resonance spectroscopy studies revealed that the ferrous center of desulfoferrodoxin could specifically and efficiently reduce O-(2), with a rate constant of 6-7 x 10(8) M(-1) s(-1). In addition, we showed that membrane and cytoplasmic E. coli protein extracts, using NADH and NADPH as electron donors, could reduce the O-(2) oxidized form of desulfoferrodoxin. Taken together, these results strongly suggest that desulfoferrodoxin behaves as a superoxide reductase enzyme and thus provide new insights into the biological mechanisms designed for protection from oxidative stresses

Non-heme iron hydroperoxo species in superoxide reductase as a catalyst for oxidation reactions

International audienceThe non-hemehigh-spin ferric iron hydroperoxo species formed in superoxide reductase catalyzesoxidative aldehyde deformylation through its nucleophile character.This species also acts as an electrophile to catalyze oxygen atom transfer in sulfoxidation reactions, highlighting the oxidation potential of non-heme iron hydroperoxo species. The mechanisms of oxygen activation and oxidation reactions catalyzed by metalloenzymes have been thoroughly investigated during the last past decades. 1-5 For cytochrome P450 5, 6 and several non-heme iron monooxygenases, 4, 7, 8 it is now well admitted that high-valent iron-oxo species formed at their active site is the effective oxidant for organic substrate oxidation and oxygen transfer. Nevertheless, the fact that alternative species, e.g. ferric iron (hydro)peroxide intermediate, 1 or other metal-oxidant adducts, 9

Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin: identification of intermediates.

International audienceFlavin reductase catalyzes the reduction of free flavins by NAD(P)H. As isolated, Escherichia coli flavin reductase does not contain any flavin prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to oxidoreduction. The reduction of riboflavin by NADPH catalyzed by flavin reductase has been studied by static and rapid kinetics absorption spectroscopies. Static absorption spectroscopy experiments revealed that, in the presence of riboflavin and reduced pyridine nucleotide, flavin reductase stabilizes, although to a small extent, a charge-transfer complex of NADP+ and reduced riboflavin. In addition, reduction of riboflavin was found to be essentially irreversible. Rapid kinetics absorption spectroscopy studies demonstrated the occurrence of two intermediates with long-wavelength absorption during the catalytic cycle. Such intermediate species exhibit spectroscopic properties similar to those of charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin and NAD(P)+, respectively, which have been identified as intermediates during the reaction of flavoenzymes of the ferredoxin-NADP+ reductase family. Thus, a minimal kinetic scheme for the reaction of flavin reductase with NADPH and riboflavin can be proposed. After formation of the Michaelis complex of flavin reductase with NADPH and riboflavin, a first intermediate, identified as a charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a second charge-transfer intermediate of enzyme-bound NADP+ and reduced riboflavin. The latter decays, yielding the Michaelis complex of flavin reductase with NADP+ and reduced riboflavin, which then dissociates to complete the reaction. These results support the initial hypothesis of a structural similarity between flavin reductase and the enzymes of the ferredoxin-NADP+ reductase family and extend it at a functional level

Superoxide reductase from Desulfoarculus baarsii: reaction mechanism and role of glutamate 47 and lysine 48 in catalysis.

International audienceSuperoxide 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