Conformational H-bonding modulation of the iron active site cysteine ligand of superoxide reductase: absorption and resonance Raman studies.

Superoxide reductases (SORs) are mononuclear non-heme iron enzymes involved in superoxide radical detoxification in some microorganisms. Their atypical active site is made of an iron atom pentacoordinated by four equatorial nitrogen atoms from histidine residues and one axial sulfur atom from a cysteinate residue, which plays a central role in catalysis. In most SORs, the residue immediately following the cysteinate ligand is an asparagine, which belongs to the second coordination sphere and is expected to have a critical influence on the properties of the active site. In this work, in order to investigate the role of this asparagine residue in the Desulfoarculus baarsii enzyme (Asn117), we carried out, in comparison with the wild-type enzyme, absorption and resonance Raman (RR) studies on a SOR mutant in which Asn117 was changed into an alanine. RR analysis was developed in order to assign the different bands using excitation in the (Cys116)-S-→ Fe3+ charge transfer band. By investigating the correlation between the (Cys116)-S-→ Fe3+ charge transfer band maximum with the frequency of each RR band in different SOR forms, we assessed the contribution of the ν(Fe-S) vibration among the different RR bands. The data showed that Asn117, by making hydrogen bond interactions with Lys74 and Tyr76, allows a rigidification of the backbone of the Cys116 ligand, as well as that of the neighboring residues Ile118 and His119. Such a structural role of Asn117 has a deep impact on the S-Fe bond. It results in a tight control of the H-bond distance between the Ile118 and His119 NH peptidic moiety with the cysteine sulfur ligand, which in turn enables fine-tuning of the S-Fe bond strength, an essential property for the SOR active site. This study illustrates the intricate roles of second coordination sphere residues to adjust the ligand to metal bond properties in the active site of metalloenzymes.

Study of a phosphorescent cationic iridium(III) complex displaying a blue-shift in crystals.

We report the synthesis and the characterization of a new cationic iridium(III) complex featuring two 1-(p-methoxyphenyl)-5-methoxybenzimidazole cyclometallating ligands and a dimethylbipyridine ancillary ligand. The complex has been fully characterized by 1D and 2D NMR (1H, 13C, 19F and 31P), elemental analysis and high-resolution mass spectrometry (HRMS). The photoluminescence studies performed in a solution, on amorphous powder and on crystals revealed an unexpected behavior. Indeed, the emission spectra observed in both solution (CH2Cl2) and amorphous powder samples are centered at around 580 nm, whereas in crystals the emission displays a large hypsochromic shift of ∼800 cm-1 (λem = 558 nm). X-ray diffraction experiments, photophysical studies and DFT calculations allow for rationalizing the hypsochromic shift.

New Acridine-Based Tridentate Ligand for Ruthenium(II): Coordination with a Twist.

A new tridentate ligand based on acridine has been synthetized. The central acridine heterocycle bears two pyridine coordinating units at positions 4 and 5. The terdentate 2,7-di- tert-butyl-4,5-di(pyridin-2-yl)acridine (dtdpa) was then coordinated to a ruthenium(II) cation. The corresponding homoleptic complex could only be obtained where both ligands coordinate to the ruthenium in a fac fashion. Thus, a heteroleptic compound (2) was constructed in combination with a terpyridine ligand in order to constrain the ligand to adopt a mer geometry. Such a coordination imposes a dramatic twist on the acridine heterocycle, resulting in an unexpected photophysical behavior. The electrochemical and photophysical properties of both complexes were studied, and the molecular structure of 2 was determined by X-ray diffraction. The two compounds absorb at low energy wavelengths, and a very weak luminescence is detected only for complex 2 in the near-infrared region.

Probing the reactivity of singlet oxygen with purines.

The reaction of singlet molecular oxygen with purine DNA bases is investigated by computational means. We support the formation of a transient endoperoxide for guanine and by classical molecular dynamics simulations we demonstrate that the formation of this adduct does not affect the B-helicity. We thus identify the guanine endoperoxide as a key intermediate, confirming a low-temperature nuclear magnetic resonance proof of its existence, and we delineate its degradation pathway, tracing back the preferential formation of 8-oxoguanine versus spiro-derivates in B-DNA. Finally, the latter oxidized 8-oxodGuo product exhibits an almost barrierless reaction profile, and hence is found, coherently with experience, to be much more reactive than guanine itself. On the contrary, in agreement with experimental observations, singlet-oxygen reactivity onto adenine is kinetically blocked by a higher energy transition state.

Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C3 methylthiolation of the D89 residue in the ribosomal S12 protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS- ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

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

Superoxide reductase (SOR) is a non-heme iron metalloenzyme that detoxifies superoxide radical in microorganisms. Its active site consists of an unusual non-heme Fe(2+) center in a [His4Cys1] 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 hydrogen bonds involving the sulfur ligand (Desulfoarculus baarsii SOR numbering). To investigate the catalytic role of these hydrogen 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 Fe(3+)-S(Cys) and S-Cß(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 involving 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 pK a of the first reaction intermediate, recently proposed to be an Fe(2+)-O2 (•-) species. These data were supported by density functional theory calculations conducted on three models of the Fe(2+)-O2 (•-) intermediate, with one, two, or no hydrogen bonds involving 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 Fe(2+)-O2 (•-) reaction intermediate to form an Fe(3+)-OOH species.

Unsymmetrical binding modes of the HOPNO inhibitor of tyrosinase: from model complexes to the enzyme.

The deciphering of the binding mode of tyrosinase (Ty) inhibitors is essential to understand how to regulate the tyrosinase activity. In this paper, by combining experimental and theoretical methods, we studied an unsymmetrical tyrosinase functional model and its interaction with 2-hydroxypyridine-N-oxide (HOPNO), a new and efficient competitive inhibitor for bacterial Ty. The tyrosinase model was a dinuclear copper complex bridged by a chelated ring with two different complexing arms (namely (bis(2-ethylpyridyl)amino)methyl and (bis(2-methylpyridyl)amino)methyl). The geometrical asymmetry of the complex induces an unsymmetrical binding of HOPNO. Comparisons have been made with the binding modes obtained on similar symmetrical complexes. Finally, by using quantum mechanics/molecular mechanics (QM/MM) calculations, we studied the binding mode in tyrosinase from a bacterial source. A new unsymmetrical binding mode was obtained, which was linked to the second coordination sphere of the enzyme.

4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

Control of the evolution of iron peroxide intermediate in superoxide reductase from Desulfoarculus baarsii. Involvement of lysine 48 in protonation.

Superoxide 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).

A fragment-based drug discovery strategy applied to the identification of NDM-1 ß-lactamase inhibitors.

Hydrolysis of ß-lactam drugs, a major class of antibiotics, by serine or metallo-ß-lactamases (SBL or MBL) is one of the main mechanisms for antibiotic resistance. New Delhi Metallo-ß-lactamase-1 (NDM-1), an acquired metallo-carbapenemase first reported in 2009, is currently considered one of the most clinically relevant targets for the development of ß-lactam-ß-lactamase inhibitor combinations active on NDM-producing clinical isolates. Identification of scaffolds that could be further rationally pharmacomodulated to design new and efficient NDM-1 inhibitors is thus urgently needed. Fragment-based drug discovery (FBDD) has become of great interest for the development of new drugs for the past few years and combination of several FBDD strategies, such as virtual and NMR screening, can reduce the drawbacks of each of them independently. Our methodology starting from a high throughput virtual screening on NDM-1 of a large library (more than 700,000 compounds) allowed, after slicing the hit molecules into fragments, to build a targeted library. These hit fragments were included in an in-house untargeted library fragments that was screened by Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR). 37 fragments were finally identified and used to establish a pharmacophore. 10 molecules based on these hit fragments were synthesized to validate our strategy. Indenone 89 that combined two identified fragments shows an inhibitory activity on NDM-1 with a Ki value of 4 µM.

Ligand-Promoted Surface Solubilization of TiO2 Nanoparticles by the Enterobactin Siderophore in Biological Medium.

Titanium dioxide nanoparticles (TiO2-NPs) are increasingly used in consumer products for their particular properties. Even though TiO2 is considered chemically stable and insoluble, studying their behavior in biological environments is of great importance to figure their potential dissolution and transformation. The interaction between TiO2-NPs with different sizes and crystallographic forms (anatase and rutile) and the strong chelating enterobactin (ent) siderophore was investigated to look at a possible dissolution. For the first time, direct evidence of anatase TiO2-NP surface dissolution or solubilization (i.e., the removal of Ti atoms located at the surface) in a biological medium by this siderophore was shown and the progressive formation of a hexacoordinated titanium-enterobactin (Ti-ent) complex observed. This complex was characterized by UV-visible and Fourier transform infrared (FTIR) spectroscopy (both supported by Density Functional Theory calculations) as well as electrospray ionization mass spectrometry (ESI-MS) and X-ray photoelectron spectroscopy (XPS). A maximum of ca. 6.3% of Ti surface atoms were found to be solubilized after 24 h of incubation, releasing Ti-ent complexes in the micromolar range that could then be taken up by bacteria in an iron-depleted medium. From a health and environmental point of view, the effects associated to the solubilization of the E171 TiO2 food additive in the presence of enterobactin and the entrance of the Ti-enterobactin complex in bacteria were questioned.

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster.

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.

NR transfer reactivity of azo-compound I of P450. How does the nitrogen substituent tune the reactivity of the species toward C-H and C=C activation?

We studied electronic structures and reactivity patterns of azo-compound I species (RN-Cpd I) by comparison to O-Cpd I of, e.g., cytochrome P450. The study shows that the RN-Cpd I species are capable of C=C aziridination and C-H amidation, in a two-state mechanism similar to that of O-Cpd I. However, unlike O-Cpd I, here the nitrogen substituent (R) exerts a major impact on structure and reactivity. Thus, it is demonstrated that Fe=NR bonds of RN-Cpd I will generally be substantially longer than Fe=O bonds; electron-withdrawing R groups will generate a very long Fe=N bond, whereas electron-releasing R groups should have the opposite effect and hence a shorter Fe=N bond. The R substituent controls also the reactivity of RN-Cpd I toward C=C and C-H bonds by exerting steric and electronic effects. Our analysis shows that an electron-releasing substituent will lower the barriers for both bond activation reactions, since the electronic factor makes the reactions highly exothermic, while an electron-withdrawing one should raise both barriers. The steric bulk of the substituent is predicted to inhibit more strongly the aziridination reactions. It is predicted that electron-releasing substituents with small bulk will create powerful aziridination reagents, whereas electron-withdrawing substituents like MeSO(2) will prefer C-H bond activation with preference that increases with steric bulk. Finally, the study predicts (i) that the reactions of RN-Cpd I will be less stereospecific than those of O-Cpd I and (ii) that aziridination will be more stereoselective than amidation.

Iron Hydroperoxide Intermediate in Superoxide Reductase: Protonation or Dissociation First? MM Dynamics and QM/MM Metadynamics Study.

Superoxide reductase is a mononuclear iron enzyme involved in superoxide radical detoxification in some bacteria. Its catalytic mechanism is associated with the remarkable formation of a ferric hydroperoxide Fe3+-OOH intermediate, which is specifically protonated on its proximal oxygen to generate the reaction product H2O2. Here, we present a computational study of the protonation mechanism of the Fe3+-OOH intermediate, at different levels of theory. This was performed on the whole system (solvated protein) using well-tempered metadynamics at the QM/MM (B3LYP/AmberFF99SB) level. Enabled by the development of a new set of force field parameters for the active site, a conformational MM study of the Fe3+-OOH species gave insights into its solvation pattern, in addition to generating the two starting conformations for the ab initio metadynamics setup. Two different protonation mechanisms for the Fe3+-OOH intermediate have been found depending on the starting structure. Whereas a possible mechanism involves at first the protonation of the hydroperoxide ligand and then dissociation of H2O2, the most probable one starts with an unexpected dissociation of the HOO- ligand from the iron, followed by its protonation. This favored reactivity was specifically linked to the influence of both the nearby conserved lysine 48 residue and the microsolvatation on the charge distribution of the oxygens of the HOO- ligand. These data highlight the crucial role of the whole environment, solvent, and protein, to describe accurately this second protonation step in superoxide reductase. This is clearly not possible with smaller models unable to reproduce correctly the mechanistically determinant charge distribution.

Formation of the active species of cytochrome p450 by using iodosylbenzene: a case for spin-selective reactivity.

The generation of the active species for the enzyme cytochrome P450 by using the highly versatile oxygen surrogate iodosylbenzene (PhIO) often produces different results compared with the native route, in which the active species is generated through O(2) uptake and reduction by NADPH. One of these differences that is addressed here is the deuterium kinetic isotope effect (KIE) jump observed during N-dealkylation of N,N-dimethylaniline (DMA) by P450, when the reaction conditions change from the native to the PhIO route. The paper presents a theoretical analysis targeted to elucidate the mechanism of the reaction of PhIO with heme, to form the high-valent iron-oxo species Compound I (Cpd I), and define the origins of the KIE jump in the reaction of Cpd I with DMA. It is concluded that the likely origin of the KIE jump is the spin-selective chemistry of the enzyme cytochrome P450 under different preparation procedures. In the native route, the reaction proceeds via the doublet spin state of Cpd I and leads to a low KIE value. PhIO, however, diverts the reaction to the quartet spin state of Cpd I, which leads to the observed high KIE values. The KIE jump is reproduced here experimentally for the dealkylation of N,N-dimethyl-4-(methylthio)aniline, by using intra-molecular KIE measurements that avoid kinetic complexities. The effect of PhIO is compared with N,N-dimethylaniline-N-oxide (DMAO), which acts both as the oxygen donor and the substrate and leads to the same KIE values as the native route.

On the suitability of strictly localized orbitals for hybrid QM/MM calculations.

In the QM/MM method we have developed (LSCF/MM), the QM and the MM parts are held together by means of strictly localized bonding orbitals (SLBOs). Generally these SLBOs are derived from localized bond orbitals (LBOs) that undergo tails deletion, resulting in a nonpredictable change of their properties. An alternative set of SLBOs is provided by the extremely localized molecular orbitals (ELMOs) approach, where the orbitals are rigorously localized on some prefixed atoms without tails on the other atoms of the molecule. A comparative study of SLBOs arising from various localization schemes and ELMOs is presented to test the reliability and the transferability of these functions within the Local Self-Consistent Field (LSCF) framework. Two types of chemical bonds were considered: C--C and C--O single bonds. The localized functions are obtained on the ethane and the methanol molecules, and are tested on beta-alanine and diethyl ether molecules. Moreover, the various protonation forms of beta-alanine have been investigated to illustrate how well the polarity variation of the chemical bond can be handled throughout a chemical process. At last, rotation energy profiles around C--C and C--O bonds are reproduced for butane and fluoromethanol. Energetic, geometric, as well as electronic factors all indicate that ELMO functions are much more transferable from one molecule to another, leading to results closer to the usual SCF reference than any other calculations involving any other localized orbitals. When the shape of the orbital is the most important factor then ELMO functions will perform as well as any other localized orbital.