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

The farnesyl transferase inhibitor RPR-130401 does not alter radiation susceptibility in human tumor cells with a K-Ras mutation in spite of large changes in ploidy and lamin B distribution

BACKGROUND: Growth inhibition by RPR-130401, a non-peptidomimetic farnesyltransferase inhibitor, was investigated without or with combined exposure to ionizing radiation in three human tumor cell lines (HCT-116, MiAPaCa-2 and A-549) bearing a point mutation in the K-Ras gene. RESULTS: RPR-130401 inhibited cell growth with an IC(50) of 50 nM (HCT-116), 120 nM (MiAPaCa-2) and 710 nM (A-549), with a poor incidence of apoptosis. The drug brought about G1 and S phase depletion together with arrest of cells in G2 phase and induced a significant accumulation of hyperploid cells showing active S phase DNA synthesis, with HCT-116 and A-549 cells being the most and least responsive, respectively. The drug also produced dramatic changes of the nuclear lamin B pattern, without lamin B cleavage and perturbation of the actin cytoskeleton. On the other hand, RPR-130401 elicited strictly additive interaction in combined treatment with ionizing radiation with regard to cell kill, altered cell cycle progression and induced hyperploidy. CONCLUSIONS: The data suggest that disruption of orderly progression through mitosis and cytokinesis, is a major outcome of drug action and that this effect proceeds from inhibition of lamin B farnesylation. It is anticipated from the strict additivity of RPR-130401 and radiation that neither induced radiation resistance nor acute or late complications of radiotherapy, should occur in combined treatment with RPR-130401

Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism.

International audienceSuperoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species

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)

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

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels

ETUDE DES MECANISMES IMPLIQUES DANS LA REPONSE CELLULAIRE PRECOCE AUX RADIATIONS IONISANTES (DOCTORAT (RADIOBIOLOGIE))

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Effets d'un inhibiteur de la poly (ADP-ribose) polymérase 1 (PARP-1) sur la réparation des cassures double-brin de l'ADN et la létalité cellulaire radio-induite en phase S

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF