Vincent Nivière

Reaction of the Desulfoferrodoxin from Desulfoarculus baarsii with Superoxide Anion EVIDENCE FOR A SUPEROXIDE REDUCTASE ACTIVITY

Desulfoferrodoxin 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 deficientEscherichia 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 × 108 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⨪2oxidized 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.

Structure of superoxide reductase bound to ferrocyanide and active site expansion upon X-ray-induced photo-reduction.

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site.

Identification of iron(III) peroxo species in the active site of the superoxide reductase SOR from Desulfoarculus baarsii.

The active site of superoxide reductase SOR consists of an Fe2+ center in an unusual [His4 Cys1] square-pyramidal geometry. It specifically reduces superoxide to produce H2O2. Here, we have reacted the SOR from Desulfoarculus baarsii directly with H2O2. We have found that its active site can transiently stabilize an Fe3+-peroxo species that we have spectroscopically characterized by resonance Raman. The mutation of the strictly conserved Glu47 into alanine results in a stabilization of this Fe3+-peroxo species, when compared to the wild-type form. These data support the hypothesis that the reaction of SOR proceeds through such a Fe3+-peroxo intermediate. This also suggests that Glu47 might serve to help H2O2 release during the reaction with superoxide.

Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O2. This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein·protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD+ from ActVB and was strongly affected by NAD+ concentration, with an IC50 of 40 μm. This control of reduced FMN transfer by NAD+ was associated with the formation of a strong charge·transfer complex between NAD+ and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

A Two-component Flavin-dependent Monooxygenase Involved in Actinorhodin Biosynthesis in Streptomyces coelicolor

The two-component flavin-dependent monooxygenases belong to an emerging class of enzymes involved in oxidation reactions in a number of metabolic and biosynthetic pathways in microorganisms. One component is a NAD(P)H:flavin oxidoreductase, which provides a reduced flavin to the second component, the proper monooxygenase. There, the reduced flavin activates molecular oxygen for substrate oxidation. Here, we study the flavin reductase ActVB and ActVA-ORF5 gene product, both reported to be involved in the last step of biosynthesis of the natural antibiotic actinorhodin in Streptomyces coelicolor. For the first time we show that ActVA-ORF5 is a FMN-dependent monooxygenase that together with the help of the flavin reductase ActVB catalyzes the oxidation reaction. The mechanism of the transfer of reduced FMN between ActVB and ActVA-ORF5 has been investigated. Dissociation constant values for oxidized and reduced flavin (FMNox and FMNred) with regard to ActVB and ActVA-ORF5 have been determined. The data clearly demonstrate a thermodynamic transfer of FMNred from ActVB to ActVA-ORF5 without involving a particular interaction between the two protein components. In full agreement with these data, we propose a reaction mechanism in which FMNox binds to ActVB, where it is reduced, and the resulting FMNred moves to ActVA-ORF5, where it reacts with O2 to generate a flavinperoxide intermediate. A direct spectroscopic evidence for the formation of such species within ActVA-ORF5 is reported.

Discovery of superoxide reductase: an historical perspective

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.

An Aromatic Hydroxylation Reaction Catalyzed by a Two-component FMN-dependent Monooxygenase THE ActVA-ActVB SYSTEM FROM STREPTOMYCES COELICOLOR

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.

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

Desulfovibrio strain SPSN was isolated from an anaerobic industrial fermenter fed with waste water from the alcohol industry. The isolate was a gram-negative, non-spore-forming, curved organism, the motility of which is provided by a single polar flagellum. The oxidation of substrates was incomplete and included glycerol and 1,3-propanediol. Sulfate, sulfite, thiosulfate, and sulfur were utilized as electron acceptors. Pyruvate, fumarate and malate could be fermented. The DNA base composition was 64.5±0.3% G+C. Cytochrome c3 and desulfoviridin were present. On the basis of these characteristics and because strain SPSN could not be ascribed to any of the existing species, the isolate is established as a new species of the genus Desulfovibrio, and the name Desulfovibrio alcoholovorans is proposed.

Pulse radiolysis studies on superoxide reductase from Treponema pallidum.

Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2 − to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi‐molecular reaction of the ferrous center with O2 −, with a rate constant of 6×108 M−1 s−1. A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s−1. This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron‐peroxide species.

The NAD(P)H:Flavin Oxidoreductase from Escherichia coli EVIDENCE FOR A NEW MODE OF BINDING FOR REDUCED PYRIDINE NUCLEOTIDES

The NAD(P)H:flavin oxidoreductase fromEscherichia coli, named Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins using NADPH or NADH as electron donor. The enzyme does not contain any prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin in a ternary complex prior to oxidoreduction. The specificity of the flavin reductase for the pyridine nucleotide was studied by steady-state kinetics using a variety of NADP analogs. Both the nicotinamide ring and the adenosine part of the substrate molecule have been found to be important for binding to the polypeptide chain. However, in the case of NADPH, the 2′-phosphate group destabilized almost completely the interaction with the adenosine moiety. Moreover, NADPH and NMNH are very good substrates for the flavin reductase, and we have shown that both these molecules bind to the enzyme almost exclusively by the nicotinamide ring. This provides evidence that the flavin reductase exhibits a unique mode for recognition of the reduced pyridine nucleotide. In addition, we have shown that the flavin reductase selectively transfers the pro-R hydrogen from the C-4 position of the nicotinamide ring and is therefore classified as an A-side-specific enzyme.