Jérôme Santolini

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

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Publications (30)101.63 Total impact

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    ABSTRACT: Nitric Oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho-physiological roles at the interface of oxidative stress and signaling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here we report an original approach that combines fast-kinetics techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterize FeII-O2 reaction intermediates in the presence of L-arginine (Arg), alkyl- and aryl-guanidines. The presence of new reaction intermediates, such as ferric heme-peroxide, that was formerly postulated, was tracked by analyzing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor but indirectly intervenes in oxygen activation mechanism by modulating the distal H-bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (Nω-hydroxy-L-arginine oxidation).
    Preview · Article · Jan 2016 · FEBS Open Bio

  • No preview · Article · Nov 2014 · Nitric Oxide
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    Jérôme Santolini · Amandine Maréchal · Alain Boussac · Pierre Dorlet
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    ABSTRACT: Nitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form.
    Full-text · Article · Sep 2013 · ChemBioChem
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    ABSTRACT: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that also functions in transcriptional regulation, oxidative stress, vesicular trafficking, and apoptosis. Because GAPDH is required for the insertion of cellular heme into inducible nitric oxide synthase [Chakravarti, R., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 18004-18009], we extensively characterized the heme binding properties of GAPDH. Substoichiometric amounts of ferric heme bound to GAPDH (one heme per GAPDH tetramer) to form a low-spin complex with UV-visible maxima at 362, 418, and 537 nm and when reduced to ferrous gave maxima at 424, 527, and 559 nm. Ferric heme association and dissociation rate constants at 10 °C were as follows: k(on) = 17800 M(-1) s(-1), k(off1) = 7.0 × 10(-3) s(-1), and k(off2) = 3.3 × 10(-4) s(-1) (giving approximate affinities of 19-390 nM). Ferrous heme bound more poorly to GAPDH and dissociated with a k(off) of 4.2 × 10(-3) s(-1). Magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data on the ferric, ferrous, and ferrous-CO complexes of GAPDH showed that the heme is bis-ligated with His as the proximal ligand. The distal ligand in the ferric complex was not displaced by CN(-) or N(3)(-) but in the ferrous complex could be displaced by CO at a rate of 1.75 s(-1) (for >0.2 mM CO). Studies with heme analogues revealed selectivity toward the coordinating metal and porphyrin ring structure. The GAPDH-heme complex was isolated from bacteria induced to express rabbit GAPDH in the presence of δ-aminolevulinic acid. Our finding of heme binding to GAPDH expands the protein's potential roles. The strength, selectivity, reversibility, and redox sensitivity of heme binding to GAPDH are consistent with it performing heme sensing or heme chaperone-like functions in cells.
    Full-text · Article · Sep 2012 · Biochemistry
  • Albane Brunel · Jérôme Santolini · Pierre Dorlet
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    ABSTRACT: H(4)B is an essential catalytic cofactor of the mNOSs. It acts as an electron donor and activates the ferrous heme-oxygen complex intermediate during Arg oxidation (first step) and NOHA oxidation (second step) leading to nitric oxide and citrulline as final products. However, its role as a proton donor is still debated. Furthermore, its exact involvement has never been explored for other NOSs such as NOS-like proteins from bacteria. This article proposes a comparative study of the role of H(4)B between iNOS and bsNOS. In this work, we have used freeze-quench to stop the arginine and NOHA oxidation reactions and trap reaction intermediates. We have characterized these intermediates using multifrequency electron paramagnetic resonance. For the first time, to our knowledge, we report a radical formation for a nonmammalian NOS. The results indicate that bsNOS, like iNOS, has the capacity to generate a pterin radical during Arg oxidation. Our current electron paramagnetic resonance data suggest that this radical is protonated indicating that H(4)B may not transfer any proton. In the 2nd step, the radical trapped for iNOS is also suggested to be protonated as in the 1st step, whereas it was not possible to trap a radical for the bsNOS 2nd step. Our data highlight potential differences for the catalytic mechanism of NOHA oxidation between mammalian and bacterial NOSs.
    No preview · Article · Jul 2012 · Biophysical Journal
  • Jérôme Santolini · Albane Brunel · Pierre Dorlet
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    ABSTRACT: NO-synthases (NOSs) produce NO by a two-oxidation reaction that sequentially converts arginine (l-Arg) into NOHA and citrulline, with NO as byproduct. Like cytochromes P450, the molecular mechanism of oxygen activation by NOS relies on the sequential transfer of two electrons and two protons, leading to the formation of a Compound-I like species that will eventually achieve l-Arg oxidation. In the last ten years, intense investigations on electron transfer in NOS have revealed that the second electron donor is a pterin cofactor, H4B. However, several questions concerning electron transfer in the NO-producing step (NOHA oxidation) remains to be addressed: the number of electron transfer (one vs two), the nature of the oxidative species (peroxo vs oxoferryl intermediate) and the electron restitution sequence. Additionally, the molecular mechanism of the numerous bacterial NOS-like proteins has been scarcely examined, with no valuable information on the electron transfer processes (number of electron, nature and role of the pterin cofactor, mechanism of NOHA oxidation…). To answer this series of questions, we have launched an investigation of electron transfer in NOS, focusing on the role of H4B. We have achieved a comparative analysis of a mammalian NOS (iNOS) and a bacterial NOS-like protein (bsNOS), for the first and second catalytic steps, by coupling Freeze-Quench to X-band and High-Field EPR spectroscopies. Our results show differences between iNOS and bsNOS, especially for the second catalytic step, that question the designation of bacterial NOS-like proteins as genuine NO synthases.
    No preview · Article · Jul 2012 · Nitric Oxide
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    ABSTRACT: Nitric oxide synthases (NOSs) are hemoproteins responsible for the biosynthesis of NO in mammals. They catalyze two successive oxidation reactions. The mechanism of oxygen activation is based on the transfer of two electrons and two protons. Despite structural analogies with cytochromes P450, the molecular mechanism of NOS remains yet to be elucidated. Because of extremely high reaction rates, conventional kinetics methods failed to trap and characterize the major reaction intermediates. Cryo-reduction methods offer a possibility to circumvent this technological lock, by triggering oxygen activation at cryogenic temperatures by using water radiolysis. However, this method is not adapted to the NOS mechanism because of the high instability of the initial Fe(II)O(2) complex (extremely fast autoxidation and/or reaction with the cofactor H(4)B). This imposed a protocol with a stable Fe(II)O(2) complex (observed only for one NOS-like protein) and that excludes any redox role for H(4)B. A relevant approach to the NOS mechanism would use H(4)B to provide the (second) electron involved in oxygen activation; water radiolysis would thus provide the first electron (heme reduction). In this context, we report here an investigation of the first electron transfer by this alternative approach, i.e., the reduction of native NOS by water radiolysis. We combined EPR and resonance Raman spectroscopies to analyze NOS reduction for a combination of different substrates, cofactor, and oxygen concentrations, and for different NOS isoforms. Our results show that cryo-reduction of native NOS is achieved for all conditions that are relevant to the investigation of the NOS mechanism.
    No preview · Article · Apr 2012 · The Journal of Physical Chemistry B
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    ABSTRACT: NO synthase enzymes (NOS) support unique single-electron transitions of a bound H(4)B cofactor during catalysis. Previous studies showed that both the pterin structure and surrounding protein residues impact H(4)B redox function during catalysis. A conserved Arg residue (Arg375 in iNOS) forms hydrogen bonds with the H(4)B ring. In order to understand the role of this residue in modulating the function of H(4)B and overall NO synthesis of the enzyme, we generated and characterized three mutants R375D, R375K and R375N of the oxygenase domain of inducible NOS (iNOSoxy). The mutations affected the dimer stability of iNOSoxy and its binding affinity toward substrates and H(4)B to varying degrees. Optical spectra of the ferric, ferrous, ferrous dioxy, ferrous-NO, ferric-NO, and ferrous-CO forms of each mutant were similar to the wild-type. However, mutants displayed somewhat lower heme midpoint potentials and faster ferrous heme-NO complex reactivity with O(2). Unlike the wild-type protein, mutants could not oxidize NOHA to nitrite in a H(2)O(2)-driven reaction. Mutation could potentially change the ferrous dioxy decay rate, H(4)B radical formation rate, and the amount of the Arg hydroxylation during single turnover Arg hydroxylation reaction. All mutants were able to form heterodimers with the iNOS G450A full-length protein and displayed lower NO synthesis activities and uncoupled NADPH consumption. We conclude that the conserved residue Arg375 (1) regulates the tempo and extent of the electron transfer between H(4)B and ferrous dioxy species and (2) controls the reactivity of the heme-based oxidant formed after electron transfer from H(4)B during steady state NO synthesis and H(2)O(2)-driven NOHA oxidation. Thus, Arg375 modulates the redox function of H(4)B and is important in controlling the catalytic function of NOS enzymes.
    Full-text · Article · Nov 2011 · Journal of inorganic biochemistry
  • Jérôme Lang · Jérôme Santolini · Manon Couture
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    ABSTRACT: Residues surrounding and interacting with the heme proximal ligand are important for efficient catalysis by heme proteins. The nitric oxide synthases (NOSs) are thiolate-coordinated enzymes that catalyze the hydroxylation of l-Arg in the first of the two catalytic cycles needed to synthesize nitric oxide. In NOSs, the indole NH group of a conserved tryptophan [W56 of the bacterial NOS-like protein from Staphylococcus aureus (saNOS)] forms a hydrogen bond with the heme proximal cysteinate ligand. The purpose of this study was to determine the impact of increasing (W56F and W56Y variants) or decreasing (W56H variant) the electron density of the proximal cysteinate ligand on molecular oxygen (O(2)) activation using saNOS as a model. We show that the removal of the indole NH···S(-) bond for W56F and W56Y caused an increase in the electron density of the cysteinate. This was probed by the decrease of the midpoint reduction potential (E(1/2)) along with weakened σ-bonding and strengthened π-backbonding with distal ligands (CO and O(2)). On the other hand, the W56H variant showed stronger Fe-OO and Fe-CO bonds (strengthened σ-bonding) along with an elevated E(1/2), which is consistent with the formation of a strong NH···S(-) hydrogen bond from H56. We also show here that changing the electron density of the proximal thiolate controls its "push effect"; whereas the rates of both O(2) activation and autoxidation of the Fe(II)O(2) complex increase with the stronger push effect created by removing the indole NH···S(-) hydrogen bond (W56F and W56Y variants), the W56H variant showed an increased stability of the complex against autoxidation and a slower rate of O(2) activation. These results are discussed with regard to the roles played by the conserved tryptophan-cysteinate interaction in the first catalytic cycle of NOS.
    No preview · Article · Nov 2011 · Biochemistry
  • Jérôme Santolini
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    ABSTRACT: Since its discovery, nitric oxide synthase (NOS), the enzyme responsible for NO biosynthesis in mammals, has been the subject of extensive investigations regarding its catalytic and molecular mechanisms. These studies reveal the high degree of sophistication of NOS functioning and regulation. However, the precise description of the NOS molecular mechanism and in particular of the oxygen activation chemistry is still lacking. The reaction intermediates implicated in NOS catalysis continue to elude identification and the current working paradigm is increasingly contested. Consequently, the last three years has seen the emergence of several competing models. All these models propose the same global reaction scheme consisting of two successive oxidation reactions but they diverge in the details of their reaction sequence. The major discrepancies concern the number, source and characteristics of proton and electron transfer processes. As a result each model proposes distinct reaction pathways with different implied oxidative species. This review aims to examine the different experimental evidence concerning NOS proton and electron transfer events and the role played by the substrates and cofactors in these processes. The resulting discussion should provide a comparative picture of all potential models for the NOS molecular mechanism.
    No preview · Article · Feb 2011 · Journal of inorganic biochemistry
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    Albane Brunel · Adjélé Wilson · Laura Henry · Pierre Dorlet · Jérôme Santolini
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    ABSTRACT: Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions.
    Preview · Article · Feb 2011 · Journal of Biological Chemistry

  • No preview · Article · Jan 2011
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    Luciana Hannibal · Jerome Santolini · Dennis J Stuehr

    Full-text · Article · Dec 2010 · Free Radical Biology and Medicine
  • Amandine Maréchal · Tony A Mattioli · Dennis J Stuehr · Jérôme Santolini
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    ABSTRACT: Nitric oxide synthases (NOSs) are multi-domain hemothiolate proteins that are the sole source of nitric oxide (NO) in mammals. NOSs can also be a source or a sink for peroxynitrite (PN), an oxidant that is suspected to be involved in numerous physiopathological processes. In a previous study, we showed that the oxygenase domain of the inducible NOS (iNOSoxy) reacts with PN and changes its oxidative reactivity [Maréchal A, Mattioli TA, Stuehr DJ & Santolini J (2007) J Biol Chem 282, 14101-14112]. Here we report a similar analysis on two other NOS isoforms, neuronal NOS (nNOS) and a bacterial NOS-like protein (bsNOS). All NOSs accelerated PN decomposition, with accumulation of a similar heme intermediate. The kinetics of PN decomposition and heme transitions were comparable among NOSs. However, their effects on PN reactivity differ greatly. All isoforms suppressed PN two-electron oxidative activity, but iNOSoxy enhanced PN one-electron oxidation and nitration potencies, the oxygenase domain of nNOS (nNOSoxy) affected them minimally, and bsNOS abolished all PN reactivities. This led to the loss of both NOS and PN decomposition activities for nNOSoxy and iNOSoxy, which may be linked to the reported alterations in their electronic absorption spectra. Bacterial bsNOS was affected to a lesser extent by reaction with PN. We propose that these differences in PN reactivity among NOSs might arise from subtle differences in their heme pockets, and could reflect the physiological specificity of each NOS isoform, ranging from oxidative stress amplification (iNOS) to detoxification (bsNOS).
    No preview · Article · Oct 2010 · FEBS Journal

  • No preview · Article · Jan 2010
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    ABSTRACT: Nitric-oxide synthases (NOS) are highly regulated heme-thiolate enzymes that catalyze two oxidation reactions that sequentially convert the substrate L-Arg first to N(omega)-hydroxyl-L-arginine and then to L-citrulline and nitric oxide. Despite numerous investigations, the detailed molecular mechanism of NOS remains elusive and debatable. Much of the dispute in the various proposed mechanisms resides in the uncertainty concerning the number and sources of proton transfers. Although specific protonation events are key features in determining the specificity and efficiency of the two catalytic steps, little is known about the role and properties of protons from the substrate, cofactors, and H-bond network in the vicinity of the heme active site. In this study, we have investigated the role of the acidic proton from the L-Arg guanidinium moiety on the stability and reactivity of the ferrous heme-oxy complex intermediate by exploiting a series of L-Arg analogues exhibiting a wide range of guanidinium pK(a) values. Using electrochemical and vibrational spectroscopic techniques, we have analyzed the effects of the analogues on the heme, including characteristics of its proximal ligand, heme conformation, redox potential, and electrostatic properties of its distal environment. Our results indicate that the substrate guanidinium pK(a) value significantly affects the H-bond network near the heme distal pocket. Our results lead us to propose a new structural model where the properties of the guanidinium moiety finely control the proton transfer events in NOS and tune its oxidative chemistry. This model may account for the discrepancies found in previously proposed mechanisms of NOS oxidation processes.
    No preview · Article · Nov 2009 · Journal of Biological Chemistry
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    Jesús Tejero · Jérôme Santolini · Dennis J Stuehr
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    ABSTRACT: During catalysis, the heme in nitric oxide synthase (NOS) binds NO before releasing it to the environment. Oxidation of the NOS ferrous heme-NO complex by O2 is key for catalytic cycling, but the mechanism is unclear. We utilized stopped-flow methods to study the reaction of O2 with ferrous heme-NO complexes of inducible and neuronal NOS enzymes. We found that the reaction does not involve heme-NO dissociation, but instead proceeds by a rapid direct reaction of O2 with the ferrous heme-NO complex. This behavior is novel and may distinguish heme-thiolate enzymes, such as NOS, from related heme proteins.
    Full-text · Article · Sep 2009 · FEBS Journal

  • No preview · Article · Dec 2008 · Nitric Oxide
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    Amandine Maréchal · Tony A Mattioli · Dennis J Stuehr · Jérôme Santolini
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    ABSTRACT: In mammals, nitric oxide (NO) is an essential biological mediator that is exclusively synthesized by nitric-oxide synthases (NOSs). However, NOSs are also directly or indirectly responsible for the production of peroxynitrite, a well known cytotoxic agent involved in numerous pathophysiological processes. Peroxynitrite reactivity is extremely intricate and highly depends on activators such as hemoproteins. NOSs present, therefore, the unique ability to both produce and activate peroxynitrite, which confers upon them a major role in the control of peroxynitrite bioactivity. We report here the first kinetic analysis of the interaction between peroxynitrite and the oxygenase domain of inducible NOS (iNOSoxy). iNOSoxy binds peroxynitrite and accelerates its decomposition with a second order rate constant of 22 x 10(4) m(-1)s(-1) at pH 7.4. This reaction is pH-dependent and is abolished by the binding of substrate or product. Peroxynitrite activation is correlated with the observation of a new iNOS heme intermediate with specific absorption at 445 nm. iNOSoxy modifies peroxynitrite reactivity and directs it toward one-electron processes such as nitration or one-electron oxidation. Taken together our results suggest that, upon binding to iNOSoxy, peroxynitrite undergoes homolytic cleavage with build-up of an oxo-ferryl intermediate and concomitant release of a NO(2)(.) radical. Successive cycles of peroxynitrite activation were shown to lead to iNOSoxy autocatalytic nitration and inhibition. The balance between peroxynitrite activation and self-inhibition of iNOSoxy may determine the contribution of NOSs to cellular oxidative stress.
    Full-text · Article · Jun 2007 · Journal of Biological Chemistry
  • A. Marechal · Ta Mattioli · Dj Stuehr · J. Santolini

    No preview · Article · Jan 2007

Publication Stats

779 Citations
101.63 Total Impact Points


  • 2011-2013
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
    • Cea Leti
      Grenoble, Rhône-Alpes, France
  • 2012
    • Université Paris-Sud 11
      Orsay, Île-de-France, France
  • 2004-2009
    • Lerner Research Institute
      • Department of Cellular and Molecular Medicine
      Cleveland, Ohio, United States