Photochemical processes observed during the reaction of superoxide reductase from Desulfoarculus baarsii with superoxide: re-evaluation of the reaction mechanism.

International audienceSuperoxide reductase SOR is an enzyme involved in superoxide detoxification in some microorganisms. Its active site consists of a non-heme ferrous center in an unusual [Fe(NHis)(4) (SCys)(1)] square pyramidal pentacoordination that efficiently reduces superoxide into hydrogen peroxide. In previous works, the reaction mechanism of the SOR from Desulfoarculus baarsii enzyme, studied by pulse radiolysis, was shown to involve the formation of two reaction intermediates T1 and T2. However, the absorption spectrum of T2 was reported with an unusual sharp band at 625 nm, very different from that reported for other SORs. In this work, we show that the sharp band at 625 nm observed by pulse radiolysis reflects the presence of photochemical processes that occurs at the level of the transient species formed during the reaction of SOR with superoxide. These processes do not change the stoichiometry of the global reaction. These data highlight remarkable photochemical properties for these reaction intermediates, not previously suspected for iron-peroxide species formed in the SOR active site. We have reinvestigated the reaction mechanism of the SOR from D. baarsii by pulse radiolysis in the absence of these photochemical processes. The T1 and T2 intermediates now appear to have absorption spectra similar to those reported for the Archaeoglobus fulgidus SOR enzymes. Although for some enzymes of the family only one transient was reported, on the whole, the reaction mechanisms of the different SORs studied so far seem very similar, which is in agreement with the strong sequence and structure homologies of their active sites

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site.

International audienceSuperoxide reductase (SOR) is a superoxide detoxification system present in some microorganisms. Its active site consists of an unusual mononuclear iron center with an FeN4S1 coordination which catalyzes the one-electron reduction of superoxide to form hydrogen peroxide. Different classes of SORs have been described depending on the presence of an additional rubredoxin-like, desulforedoxin iron center, whose function has remained unknown until now. In this work, we investigated the mechanism of the reduction of the SOR iron active site using the NADPH:flavodoxin oxidoreductase from Escherichia coli, which was previously shown to efficiently transfer electrons to the Desulfoarculus baarsii SOR. When present, the additional rubredoxin-like iron center could function as an electronic relay between cellular reductases and the iron active site for superoxide reduction. This electron transfer was mainly intermolecular, between the rubredoxin-like iron center of one SOR and the iron active site of another SOR. These data provide the first experimental evidence for a possible role of the rubredoxin-like iron center in the superoxide detoxifying activity of SOR

Incidence of adverse events in antipsychotic-naïve children and adolescents treated with antipsychotic drugs: a French multicentre naturalistic study protocol (ETAPE)

ETAPE Study groupInternational audienceIntroduction: In France, over recent years, the prescription rate of antipsychotic (AP) remained stable in children and adolescents. Prescription of second-generation antipsychotics increased, whereas prescription of first-generation antipsychotics decreased. Off-label prescriptions are very frequent in this population. Adverse events (AEs) in youth treated with AP are common and may be severe. AEs have hitherto been poorly monitored in naturalistic studies independent from industry.Method and analysis: We describe a French prospective multicentre study in an AP-naïve paediatric population named Etude de la Tolérance des AntiPsychotique chez l'Enfant (ETAPE). The study started in April 2013. So far, 200 patients have been included. The inclusion criteria are: male or female inpatients aged from 6 to 18 years, treated with an AP drug for less than 28 days, never been treated or having received AP for less than 3 months, discontinued at least 6 months prior to inclusion. These assessments of AE are performed at inclusion, as well as at 3, 6, 9 and 12 months after the introduction of the AP. The monitoring period will end in May 2016.Ethics and dissemination: The study protocol was approved by the Ethics Committee 'Sud Méditerrané V' (number 12.082) and by the French National Agency for Medicines and Health Products Safety (number 2012-004546-15). All patients and their parents signed informed consent on enrolment in the study. We will submit the results of the study to relevant journals and offer national and international presentations. This study will enable better characterisation of the prescription of AP drugs. The results will further help to develop quality standards and recommendations for monitoring AE during the prescription of AP

Control of the Evolution of Iron Peroxide Intermediate in Superoxide Reductase from Desulfoarculus baarsii. Involvement of Lysine 48 in Protonation

International audienceSuperoxide reductase is a nonheme iron metalloenzyme that detoxifies superoxide anion radicals O(2)(•-) in some microorganisms. Its catalytic mechanism was previously proposed to involve a single ferric iron (hydro)peroxo intermediate, which is protonated to form the reaction product H(2)O(2). Here, we show by pulse radiolysis that the mutation of the well-conserved lysine 48 into isoleucine in the SOR from Desulfoarculus baarsii dramatically affects its reaction with O(2)(•-). Although the first reaction intermediate and its decay are not affected by the mutation, H(2)O(2) is no longer the reaction product. In addition, in contrast to the wild-type SOR, the lysine mutant catalyzes a two-electron oxidation of an olefin into epoxide in the presence of H(2)O(2), suggesting the formation of iron-oxo intermediate species in this mutant. In agreement with the recent X-ray structures of the peroxide intermediates trapped in a SOR crystal, these data support the involvement of lysine 48 in the specific protonation of the proximal oxygen of the peroxide intermediate to generate H(2)O(2), thus avoiding formation of iron-oxo species, as is observed in cytochrome P450. In addition, we proposed that the first reaction intermediate observed by pulse radiolysis is a ferrous-iron superoxo species, in agreement with TD-DFT calculations of the absorption spectrum of this intermediate. A new reaction scheme for the catalytical mechanism of SOR with O(2)(•-) is presented in which ferrous iron-superoxo and ferric hydroperoxide species are reaction intermediates, and the lysine 48 plays a key role in the control of the evolution of iron peroxide intermediate to form H(2)O(2)

Formation of high-valent iron-oxo species in superoxide reductase: characterization by resonance Raman spectroscopy.

International audienceSuperoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site

Hydrogen bonding to the cysteine ligand of superoxide reductase: acid–base control of the reaction intermediates

International audienceSuperoxide reductase SOR is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe2+ center in a [His4 Cys1] square pyramidal pentacoordination, with the axial cysteine ligand proposed to be an essential feature in catalysis. Two NH peptide groups from isoleucine 118 and histidine 119 establish H-bondings with the sulfur ligand (Desulfoarculus baarsii SOR numbering). In order to investigate the catalytic role of these H-bonds, the isoleucine 118 residue of the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate or serine residues. Resonance Raman spectroscopy showed that the mutations specifically induced an increase of the strength of the Fe3+-S(Cys) and S-Cβ(Cys) bonds as well as a change in conformation of the cysteinyl side chain, which was associated with the alteration of the NH hydrogen bonding to the sulfur ligand. The effects of the isoleucine mutations on the reactivity of SOR with O2●- were investigated by pulse radiolysis. These studies showed that the mutations induced a specific increase of the pKa of the first reaction intermediate, recently proposed to be an Fe2+-O2●- species. These data were supported by DFT calculations carried out on three models of the Fe2+-O2●- intermediate, with one, two or no H-bonds on the sulfur ligand. Our results demonstrated that the hydrogen bonds between the NH (peptide) and the cysteine ligand tightly control the rate of protonation of the Fe2+-O2●- reaction intermediate to form an Fe3+-OOH species

Superoxyde réductase : Mécanisme de transfert d'électrons vers le site actif et rôle de la lysine 48 dans la catalyse.

Superoxide reductase (SOR) is a metalloprotein which catalyses the reduction of the superoxide into H2O2. For its catalytic activity, SOR requires physiological partners as electron donor (cellular reductases). SORs of Class 1, such as that of Desulfoarculus baarsii, possess besides the active site, an additional iron center, rubredoxin type with an unknown function. We showed that this center plays a role of electronic relay between reductases and the active site of SOR. However, this electron transfer between the rubredoxin center and the active site is intermolecular, between two molecules of SOR. We propose that the presence of this iron center allows the SOR to adapt to various cellular reductases in order to optimize its detoxification activity. The catalytic mechanism of the SOR had been studied by rapid kinetic. We show that the previous studies on the SOR from D. baarsii were perturbed by a photochemical phenomenon. The reaction mechanism has been reinvestigated and allowed us to propose a new reaction mechanism of reduction of the superoxide by the SOR of D. baarsii. Our studies on the SOR mutant K48I of D. baarsii allowed us to bring to light the essential role of that lysine 48 in the protonation of the reaction intermediate Fe3+-OOH. In absence of this lysine, this reaction intermediate does not drive any more to the formation of the product of the reaction H2O2. Our data suggest that in absence of this lysine, the intermediate Fe3+-OOH evolves towards a iron-oxo type species, as observed in the cytochrome P450 oxygenases. So, the lysine 48 appears as an essential residue, which allows the SOR to direct the evolution of the intermediary Fe3+-OOH.La superoxyde réductase (SOR) est une métalloprotéine qui catalyse la réduction du superoxyde en H2O2. Pour son activité catalytique, la SOR nécessite des réductases cellulaires comme donneurs d'e-. Les SORs de Classe 1, telle que celle de Desulfoarculus baarsii, possèdent en plus du site actif, un centre de type rubrédoxine dont la fonction est inconnue. Nous avons montré que ce centre peut jouer un rôle de relais électronique entre les réductases et le site actif de la SOR. Cependant, ce transfert d'e- entre le centre rubrédoxine et le site actif se fait de façon intermoléculaire, entre deux molécules de SOR. Nous proposons que ce centre permet à la SOR de s'adapter à une large gamme de réductases cellulaires et optimise ainsi l'activité de détoxification du O2•- de la SOR. Le mécanisme catalytique de la SOR a été très étudié par cinétique rapide. Nous montrons que les études antérieures sur la SOR de D. baarsii ont été perturbées par un phénomène photochimique. Les études du mécanisme de la SOR ont été reprises et nous ont permis de proposer un nouveau mécanisme réactionnel de réduction du superoxyde par la SOR de D. baarsii. Nos études sur le mutant SOR K48I de D. baarsii ont permis de mettre en évidence le rôle essentiel de la lysine 48 dans la protonation de l'intermédiaire réactionnel Fe3+-OOH. En absence de cette lysine, cet intermédiaire ne conduit plus à la formation du produit de la réaction H2O2. Nos données suggèrent que l'intermédiaire Fe3+-OOH évolue alors vers une espèce de type fer-oxo, comme dans les oxygénases de type cytochrome P450. Ainsi, la lysine 48 apparaît comme un résidu essentiel, qui permet à la SOR d'orienter l'évolution de l'intermédiaire Fe3+-OOH

Superoxyde réductase : Mécanisme de transfert d'électrons vers le site actif et rôle de la lysine 48 dans la catalyse.

Superoxide reductase (SOR) is a metalloprotein which catalyses the reduction of the superoxide into H2O2. For its catalytic activity, SOR requires physiological partners as electron donor (cellular reductases). SORs of Class 1, such as that of Desulfoarculus baarsii, possess besides the active site, an additional iron center, rubredoxin type with an unknown function. We showed that this center plays a role of electronic relay between reductases and the active site of SOR. However, this electron transfer between the rubredoxin center and the active site is intermolecular, between two molecules of SOR. We propose that the presence of this iron center allows the SOR to adapt to various cellular reductases in order to optimize its detoxification activity. The catalytic mechanism of the SOR had been studied by rapid kinetic. We show that the previous studies on the SOR from D. baarsii were perturbed by a photochemical phenomenon. The reaction mechanism has been reinvestigated and allowed us to propose a new reaction mechanism of reduction of the superoxide by the SOR of D. baarsii. Our studies on the SOR mutant K48I of D. baarsii allowed us to bring to light the essential role of that lysine 48 in the protonation of the reaction intermediate Fe3+-OOH. In absence of this lysine, this reaction intermediate does not drive any more to the formation of the product of the reaction H2O2. Our data suggest that in absence of this lysine, the intermediate Fe3+-OOH evolves towards a iron-oxo type species, as observed in the cytochrome P450 oxygenases. So, the lysine 48 appears as an essential residue, which allows the SOR to direct the evolution of the intermediary Fe3+-OOH.La superoxyde réductase (SOR) est une métalloprotéine qui catalyse la réduction du superoxyde en H2O2. Pour son activité catalytique, la SOR nécessite des réductases cellulaires comme donneurs d'e-. Les SORs de Classe 1, telle que celle de Desulfoarculus baarsii, possèdent en plus du site actif, un centre de type rubrédoxine dont la fonction est inconnue. Nous avons montré que ce centre peut jouer un rôle de relais électronique entre les réductases et le site actif de la SOR. Cependant, ce transfert d'e- entre le centre rubrédoxine et le site actif se fait de façon intermoléculaire, entre deux molécules de SOR. Nous proposons que ce centre permet à la SOR de s'adapter à une large gamme de réductases cellulaires et optimise ainsi l'activité de détoxification du O2•- de la SOR. Le mécanisme catalytique de la SOR a été très étudié par cinétique rapide. Nous montrons que les études antérieures sur la SOR de D. baarsii ont été perturbées par un phénomène photochimique. Les études du mécanisme de la SOR ont été reprises et nous ont permis de proposer un nouveau mécanisme réactionnel de réduction du superoxyde par la SOR de D. baarsii. Nos études sur le mutant SOR K48I de D. baarsii ont permis de mettre en évidence le rôle essentiel de la lysine 48 dans la protonation de l'intermédiaire réactionnel Fe3+-OOH. En absence de cette lysine, cet intermédiaire ne conduit plus à la formation du produit de la réaction H2O2. Nos données suggèrent que l'intermédiaire Fe3+-OOH évolue alors vers une espèce de type fer-oxo, comme dans les oxygénases de type cytochrome P450. Ainsi, la lysine 48 apparaît comme un résidu essentiel, qui permet à la SOR d'orienter l'évolution de l'intermédiaire Fe3+-OOH

Superoxyde réductase (mécanisme de transfert d'électrons vers le site actif et rôle de la lysine 48 dans la catalyse)

La superoxyde réductase (SOR) est une métalloprotéine qui catalyse la réduction du radical superoxyde en peroxyde d'hydrogène. Son site actif est constitué d'un centre mononucléaire de fer pentacoordiné tout à fait particulier de type [FeHisN4CysS1]. Pour son activité catalytique, la SOR nécessite des partenaires physiologiques comme donneur d'électron (réductases cellulaires). Les SORs de Classe 1, telle que celle de Desulfoarculus baarsii, possèdent en plus du site actif, un centre de type rubrédoxine [Fe(SCys)4] qui ne réagit pas avec le superoxyde et dont la fonction est inconnue. Nous avons montré que ce centre rubrédoxine peut jouer un rôle de relais électronique entre les réductases et le site actif de la SOR. Cependant, ce transfert d'électron entre le centre rubrédoxine et le site actif ne se fait pas de façon intramoléculaire mais intermoléculaire, entre deux molécules de SOR. Nous proposons que la présence de ce centre permet à la SOR de s'adapter à une très large gamme de réductases cellulaires et optimise ainsi l'activité de détoxification du radical superoxyde de la SOR. Le mécanisme catalytique de la SOR a été très étudié par la technique de radiolyse pulsée. Nous montrons que les études antérieures sur la SOR de D. baarsii ont été perturbées par un phénomène photochimique, résultant d'une propriété particulière d'un des intermédiaires réactionnels du cycle catalytique. Les études du mécanisme de la SOR ont été reprises en absence de cet effet photochimique et nous ont permis de proposer un nouveau mécanisme réactionnel de réduction du superoxyde par la SOR de D. baarsii. Nos études sur le mutant SOR K48I de D. baarsii nous ont permis de mettre en évidence le rôle essentiel que joue la lysine 48 dans la protonation de l'intermédiaire réactionnel Fe3+-hydroperoxyde. En absence de cette lysine, nous avons observé une modification profonde de la réactivité de cet intermédiaire, qui ne conduit plus à la formation du produit de la réaction H2O2. Nous avons montré que l'espèce Fe3+-hydroperoxyde formée au sein de ce mutant est alors capable de réaliser des réactions d'oxydation spécifiques, telle la transformation de thioanisole en méthyl phényl sulfoxyde. Nos données suggèrent fortement qu'en absence de cette lysine, l'intermédiaire Fe3+-hydroperoxyde évolue vers une espèce à haut degré d'oxydation de type fer-oxo, fortement oxydante, responsable de ces oxydations. Ainsi, la lysine 48 apparaît comme un résidu essentiel, qui permet pour la SOR d'orienter l'évolution de l'intermédiaire Fe3+-hydroperoxyde vers la production de H2O2, plutôt que vers la formation d'entités de type fer-oxo à haut degré d'oxydation, comme le font les oxygénases de type cytochrome P450.Superoxide reductase (SOR) is a metalloprotein which catalyses the reduction of the radical superoxide into hydrogen peroxide. Its active site is constituted by a mononuclear iron center pentacoordinated [FeHisN4CysS1]. For its catalytic activity, SOR requires physiological partners as electron donor (cellular reductases). SORs of Class 1, such as that of Desulfoarculus baarsii, possess besides the active site, an additional iron center, rubredoxin [Fe(SCys)4] type, which does not react with the superoxide and with an unknown function. We showed that this rubredoxin center plays a role of electronic relay between reductases and the active site of the SOR. However, we show that this electron transfer between the rubredoxin center and the active site is not intramolecular but intermolecular, between two molecules of SOR. We propose that the presence of this iron center allows the SOR to adapt to various cellular reductases in order to optimize its detoxification activity. The catalytic mechanism of the SOR had been studied by pulsed radiolysis. We show that the previous studies on the SOR from D. baarsii were perturbed by a photochemical phenomenon, resulting from a particular property of one of the key reaction intermediates of the catalytic cycle. The reaction mechanism has been reinvestigated in the absence of this photochemical effect and allowed us to propose a new reaction mechanism of reduction of the superoxide by the SOR of D. baarsii. Our studies on the SOR mutant K48I of D. baarsii allowed us to bring to light the essential role of that lysine 48 in the protonation of the reaction intermediate Fe3+-hydroperoxyde. In absence of this lysine, we observed a modification of the reactivity of this reaction intermediate, which does not drive any more to the formation of the product of the reaction H2O2. We showed that the species Fe3+-hydroperoxyde formed within this mutant is then capable to carry out specific reactions of oxidation, such the transformation of thioanisole methyl phenyl sulfoxyde. Our data strongly suggest that in absence of this lysine, the intermediate Fe3+-hydroperoxide evolves towards a high degree oxidation species, iron-oxo type, strongly oxidizing, responsible for these oxidations. So, the lysine 48 appears as an essential residue, which allows the SOR to direct the evolution of the intermediary Fe3+-hydroperoxyde to the production of H2O2, rather than to the formation of high-valent iron-oxo species, as observed in the cytochrome P450 oxygenases.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF