Influence of thiolate ligands on reductive N-O bond activation. Probing the O2(-) binding site of a biomimetic superoxide reductase analogue and examining the proton-dependent reduction of nitrite

Nitric oxide (NO) is frequently used to probe the substrate-binding site of "spectroscopically silent" non-heme Fe(2+) sites of metalloenzymes, such as superoxide reductase (SOR). Herein we use NO to probe the superoxide binding site of our thiolate-ligated biomimetic SOR model [Fe(II)(S(Me(2))N(4)(tren))](+) (1). Like NO-bound trans-cysteinate-ligated SOR (SOR-NO), the rhombic S = 3/2 EPR signal of NO-bound cis-thiolate-ligated [Fe(S(Me(2))N(4)(tren)(NO)](+) (2; g = 4.44, 3.54, 1.97), the isotopically sensitive ν(NO)(ν((15)NO)) stretching frequency (1685(1640) cm(-1)), and the 0.05 ? decrease in Fe-S bond length are shown to be consistent with the oxidative addition of NO to Fe(II) to afford an Fe(III)-NO(-) {FeNO}(7) species containing high-spin (S = 5/2) Fe(III) antiferromagnetically coupled to NO(-) (S = 1). The cis versus trans positioning of the thiolate does not appear to influence these properties. Although it has yet to be crystallographically characterized, SOR-NO is presumed to possess a bent Fe-NO similar to that of 2 (Fe-N-O = 151.7(4)°). The N-O bond is shown to be more activated in 2 relative to N- and O-ligated {FeNO}(7) complexes, and this is attributed to the electron-donating properties of the thiolate ligand. Hydrogen-bonding to the cysteinate sulfur attenuates N-O bond activation in SOR, as shown by its higher ν(NO) frequency (1721 cm(-1)). In contrast, the ν(O-O) frequency of the SOR peroxo intermediate and its analogues is not affected by H-bonds to the cysteinate sulfur or other factors influencing the Fe-SR bond strength; these only influence the ν(Fe-O) frequency. Reactions between 1 and NO(2)(-) are shown to result in the proton-dependent heterolytic cleavage of an N-O bond. The mechanism of this reaction is proposed to involve both Fe(II)-NO(2)(-) and {FeNO}(6) intermediates similar to those implicated in the mechanism of NiR-promoted NO(2)(-) reduction.     

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.     

Sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations on superoxide reductase: role of the axial thiolate in reactivity

Superoxide reductase (SOR) is a non-heme iron enzyme that reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge X-ray absorption spectroscopy (XAS) is used to investigate the ground-state electronic structure of the resting high-spin and CN- bound low-spin FeIII forms of the 1Fe SOR from Pyrococcus furiosus. A computational model with constrained imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms), and H-bonding to the exchangeable axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin FeIII-OOH species and its subsequent protonation resulting in H2O2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the FeIII bond to the proximal oxygen of this hydroperoxo species, which raises its pKa by an additional 5 log units relative to the pKa of a primarily anionic ligand, facilitating its protonation. A comparison with cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin FeIII-OOH species that would not be capable of efficient H2O2 release due to a spin-crossing barrier associated with formation of a high-spin 5C FeIII product. Additionally, the presence of the dianionic porphyrin pi ring in cytochrome P450 allows O-O heterolysis, forming an FeIV-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C FeIII site that results from the product release at the end of the O2- reduction cycle is calculated to be capable of reacting with a second O2-, resulting in superoxide dismutase (SOD) activity. However, in contrast to FeSOD, the 5C FeIII site of SOR, which is more positively charged, is calculated to have a high affinity for binding a sixth anionic ligand, which would inhibit its SOD activity.     

The reaction mechanism of iron and manganese superoxide dismutases studied by theoretical calculations

We have studied the detailed reaction mechanism of iron and manganese superoxide dismutase with density functional calculations on realistic active-site models, with large basis sets and including solvation, zero-point, and thermal effects. The results indicate that the conversion of O2- to O2 follows an associative mechanism, with O2- directly binding to the metal, followed by the protonation of the metal-bound hydroxide ion, and the dissociation of 3O2. All these reaction steps are exergonic. Likewise, we suggest that the conversion of O2- to H2O2 follows an at least a partly second-sphere pathway. There are small differences in the preferred oxidation and spin states, as well as in the geometries, of Fe and Mn, but these differences have little influence on the energetics, and therefore on the reaction mechanism of the two types of superoxide dismutases. For example, the two metals have very similar reduction potentials in the active-site models, although they differ by 0.7 V in water solution. The reaction mechanisms and spin states seem to have been designed to avoid spin conversions or to facilitate them by employing nearly degenerate spin states.     

Steric and electronic control over the reactivity of a thiolate-ligated Fe(II) complex with dioxygen and superoxide: reversible mu-oxo dimer formation

The reactivity between a thiolate-ligated five-coordinate complex [FeII(SMe2N4(tren))]+ (1) and dioxygen is examined in order to determine if O2 activation, resembling that of the metalloenzyme cytochrome P450, can be promoted even when O2 binds cis, as opposed to trans, to a thiolate. Previous work in our group showed that [FeII(SMe2N4(tren))]+ (1) reacts readily with superoxide (O2-) in the presence of a proton source to afford H2O2 via an Fe(III)-OOH intermediate, thus providing a biomimetic model for the metalloenzyme superoxide reductase (SOR). Addition of O2 to 1 affords binuclear mu-oxo-bridged [FeIII(SMe2N4(tren))]2(mu2-O)(PF6)2.3MeCN (3). At low temperatures, in protic solvents, an intermediate is detected, the details of which will be the subject of a separate paper. Although the thiolate ligand does not appear to perturb the metrical parameters of the unsupported mu-oxo bridge (Fe-O= 1.807(8) A, and Fe-O-Fe= 155.3(5) degrees fall in the usual range), it decreases the magnetic coupling between the irons (J=-28 cm(-1)) and creates a rather basic oxo site. Protonation of this oxo using strong (HBF4, HCl) or weak (HOAc, NH4PF6, LutNHCl) acids results in bridge cleavage to cleanly afford the corresponding monomeric anion-ligated (OAc- (6), or Cl- (7)) or solvent-ligated (MeCN (4)) derivatives. Addition of OH- converts [FeIII(SMe2N4(tren))(MeCN2+ (4) back to mu-oxo 3. Thus, mu-oxo bridge cleavage is reversible. The protonated mu-hydroxo-bridged intermediate is not observed. In an attempt to prevent mu-oxo dimer formation, and facilitate the observation of O2-bound intermediates, a bulkier tertiary amine ligand, tren-Et4= N-(2-amino-ethyl)-N-(2-diethylamino-ethyl)-N',N'-diethyl-ethane-1,2-diamine, and the corresponding [FeII(SMe2N4(tren-Et4))]+ (5) complex was synthesized and structurally characterized. Steric repulsive interactions create unusually long FeII-N(3,4) amine bonds in 5 (mean distance=2.219(1) A). The [(tren-Et4)N4SMe2]1- ligand is unable to accommodate iron in the +3 oxidation state, and consequently, in contrast to most thiolate-ligated Fe(II) complexes, [FeII(SMe2N4(tren-Et4))]+ (5) does not readily react with O2. Oxidation of 5 is irreversible, and the potential (Epa=+410 mV (vs SCE)) is anodically shifted relative to 1 (E1/2=-100 mV (vs SCE)).     

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).     

Superoxide anion is the intermediate in the oxygen reduction reaction on platinum electrodes

We identified the superoxide anion as the intermediate in the oxygen (O2) reduction reaction on a platinum (Pt) electrode in alkaline solution (pH = 11) using a surface-enhanced infrared spectroscopy technique with an attenuated total reflection mode. Spectral and voltammetry data, together with the vibrational frequencies calculated using the density functional theory, provide evidence for the formation of O2-. The supporting evidence includes similar spectra that we obtained for O2 reduction on Pt in acetonitrile solutions and a lack of spectra in the absence of O2 or its reduction. The appearance of O2- means that the series reaction pathway operates during O2 reduction on Pt electrodes in alkaline solutions and very likely also in acid solutions. This finding opens up the possibility of formulating a detailed reaction mechanism on surfaces supporting a four-electron reduction, which is critical in completely understanding the kinetics of O2 reduction, thus resolving dilemmas in the theoretical treatment of its kinetics and the design of new electrocatalysts.     

A theoretical study on the reaction pathways of peroxynitrite formation and decay at nonheme iron centers

A computational study based on density functional theory was undertaken to identify possible reaction pathways for the formation and decomposition of peroxynitrite at models of the active sites of the nonheme superoxide scavenging enzymes superoxide reductase (SOR) and iron superoxide dismutase (FeSOD). Two peroxynitrite isomers and their possible protonated states were investigated, namely Fe? OONO^?, Fe? N(O)OO^?, Fe? OONOH, and Fe? N(O)OOH. Peroxynitrite formation at the active sites was assumed by either the interaction of a peroxynitrite cis/trans anion with the pentacoordinated iron active site or the interaction between a nitric oxide bound adduct and superoxide; both scenarios were found to be facile for all models investigated. The ferrous adducts of the Fe? OONO^?isomer were found to undergo instant heterolytic cleavage of the O? ONO bond to yield nitrite, whereas for the ferric adducts, the homolytic cleavage of the O? ONO bond to yield nitrogen dioxide was found to be energetically facile. For the Fe? N(O)OO^? isomer, the active site models of FeSOD and SOR were only able to accommodate the cis isomer of peroxynitrite. Ferric adducts of the cis Fe? OONO^? isomer were found to be energetically more stable than their trans counterparts and were also more stable than the cis adducts of the Fe? N(O)OO^? isomer; conversely, the protonated forms of all adducts of the Fe? OONOH isomer were found to be lower in energy than their equivalent Fe? N(O)OOH adducts. Multiple reaction pathways for the decomposition of the formed peroxynitrite adducts (whether the anions or the protonated forms) were proposed and explored. The energy requirements for the decomposition processes ranged from exothermic to highly demanding depending on the peroxynitrite isomer, the type of model (whether an SOR or FeSOD active site), and the oxidation state of iron. © 2014 Wiley Periodicals, Inc.     

Investigation of the mechanism of formation of a thiolate-ligated Fe(III)-OOH

Kinetic studies aimed at determining the most probable mechanism for the proton-dependent [Fe(II)(S(Me2)N(4)(tren))](+) (1) promoted reduction of superoxide via a thiolate-ligated hydroperoxo intermediate [Fe(III)(S(Me2)N(4)(tren))(OOH)](+) (2) are described. Rate laws are derived for three proposed mechanisms, and it is shown that they should conceivably be distinguishable by kinetics. For weak proton donors with pK(a(HA)) > pK(a(HO(2))) rates are shown to correlate with proton donor pK(a), and display first-order dependence on iron, and half-order dependence on superoxide and proton donor HA. Proton donors acidic enough to convert O(2)(-) to HO(2) (in tetrahydrofuran, THF), that is, those with pK(a(HA)) < pK(a(HO(2))), are shown to display first-order dependence on both superoxide and iron, and rates which are independent of proton donor concentration. Relative pK(a) values were determined in THF by measuring equilibrium ion pair acidity constants using established methods. Rates of hydroperoxo 2 formation displays no apparent deuterium isotope effect, and bases, such as methoxide, are shown to inhibit the formation of 2. Rate constants for p-substituted phenols are shown to correlate linearly with the Hammett substituent constants σ(-). Activation parameters ((ΔH(++) = 2.8 kcal/mol, ΔS(++) = -31 eu) are shown to be consistent with a low-barrier associative mechanism that does not involve extensive bond cleavage. Together, these data are shown to be most consistent with a mechanism involving the addition of HO(2) to 1 with concomitant oxidation of the metal ion, and reduction of superoxide (an "oxidative addition" of sorts), in the rate-determining step. Activation parameters for MeOH- (ΔH(++) = 13.2 kcal/mol and ΔS(++) = -24.3 eu), and acetic acid- (ΔH(++) = 8.3 kcal/mol and ΔS(++) = -34 eu) promoted release of H(2)O(2) to afford solvent-bound [Fe(III)(S(Me2)N(4)(tren))(OMe)](+) (3) and [Fe(III)(S(Me2)N(4)(tren))(O(H)Me)](+) (4), respectively, are shown to be more consistent with a reaction involving rate-limiting protonation of an Fe(III)-OOH, than with one involving rate-limiting O-O bond cleavage. The observed deuterium isotope effect (k(H)/k(D) = 3.1) is also consistent with this mechanism.     

What causes iron-sulphur bonds in active sites of one-iron superoxide reductase and two-iron superoxide reductase to differ?

The electronic and vibrational properties of [Fe(NHis)₄(SCys)] sites responsible for the catalysis of superoxide reduction in two types of superoxide reductase (SOR), one-iron superoxide reductase (1Fe-SOR) and two-iron superoxide reductase (2Fe-SOR), were compared previously (Clay et al., 2003); the differences between these two classes of SOR, examined by UV-VIS and NIR absorption, VTMCD, and vibrational spectroscopy techniques, were interpreted as being indicative of weaker Fe-S bonds in 2Fe-SOR in comparison with 1Fe-SOR. Here, we report on density functional (DFT) and semi-empirical (ZINDO/S-CI) calculations exploring the extent of this difference in bonding between the two classes of SOR. The differences observed experimentally between the electronic spectra of the two SORs are shown to probably arise either from different degrees of torsion between the Fe—ligand bonds or from differences in length of the Fe—carboxylate bond, but are shown to be incompatible with any significant differences in Fe—S bond lengths. The differences observed in the vibrational spectra between the two SORs are shown to correlate with differences in the Fe-S bond length of no more than 0.01 ?, which in turn arise from slight differences in the polarity of the medium surrounding the iron active site in the two proteins.     

A superoxide dismutase-modified electrode that detects superoxide ion

A superoxide dismutase (SOD)-modified electrode, in which SOD is oriented on the gold electrode via a self-assembled monolayer of cysteine so as to allow its direct electrode reaction, possesses a bi-directional electrocatalysis for both the oxidation of superoxide ion (O2-) to O2 and the reduction of O2- to H2O2 and functions as a third generation O2- biosensor.     

Mechanisms of electron transfer in catalysis by copper zinc superoxide dismutase

Activated oxygen intermediates during copper zinc superoxide dismutase (SOD) catalysis were investigated using an isotope fractionation technique and natural abundance reagents. Competitive oxygen kinetic isotope effects (KIEs) are reported for the enzyme-catalyzed disproportionation of superoxide as well as the stoichiometric reaction of reduced SOD with molecular oxygen. Analysis within the context of quantum mechanical electron transfer theory provides evidence against an outer-sphere mechanism for O2*- oxidation. A CuII-O2-I intermediate is, therefore, proposed. The SOD-catalyzed oxidation of O2*- is characterized by an inverse (<1) KIE which is similar to those determined for the analogous reactions of synthetic copper compounds. An inverse kinetic isotope effect upon the enzymatic reduction of O2*- is also observed and proposed to arise from rate-determining proton transfer which leads to the formation of HO2* in the SOD active site.     

Formation of High‐Valent Iron–Oxo Species in Superoxide Reductase: Characterization by Resonance Raman Spectroscopy

Superoxide 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.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

Mechanism of oxygen electroreduction on gold surfaces in basic media

The mechanism of the electroreduction of oxygen on Au surfaces in basic media is examined using surface-enhanced Raman scattering (SERS) measurements and density functional theory (DFT) calculations. The spectroscopy reveals superoxide species as a reduction intermediate throughout the oxygen electroreduction, while no peroxide is detected. The spectroscopy also shows the presence of superoxide after the addition of hydrogen peroxide. The calculations show no effect of OH addition to the Au(100) surface with regard to O-O length. These results suggest that the four-electron reduction of O(2) on Au(100) in base arises from a disproportionation mechanism which is enhanced on Au(100) relative to the other two low Miller index faces of Au.     

Inner-sphere mechanism for molecular oxygen reduction catalyzed by copper amine oxidases

Copper and topaquinone (TPQ) containing amine oxidases utilize O2 for the metabolism of biogenic amines while concomitantly generating H2O2 for use by the cell. The mechanism of O2 reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the Cu(I), TPQ semiquinone state. A new mechanistic proposal for O2 reduction is advanced on the basis of thermodynamic considerations together with kinetic studies (at varying pH, temperature, and viscosity), the identification of steady-state intermediates, and the analysis of competitive oxygen kinetic isotope effects, (18)O KIEs, [kcat/KM((16,16)O2)]/[kcat/KM((16,18)O2)]. The (18)O KIE = 1.0136 +/- 0.0013 at pH 7.2 is independent of temperature from 5 degrees C to 47 degrees C and insignificantly changed to 1.0122 +/- 0.0020 upon raising the pH to 9, thus indicating the absence of kinetic complexity. Using density functional methods, the effect is found to be precisely in the range expected for reversible O2 binding to Cu(I) to afford a superoxide, [Cu(II)(eta(1)-O2)(-I)](+), intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu(II)(eta(1)-O2)(-II), intermediate driving the reduction of O2. The similar (18)O KIEs reported for copper amine oxidases from other sources raise the possibility that all enzymes react by related inner-sphere mechanisms although additional experiments are needed to test this proposal.     

The trans effect in square-planar platinum(II) complexes--a density functional study

The mechanism of substitution water exchange reactions in square planar trans-Pt[(NH(3))(2)T(H(2)O)](n+) complexes is studied (T = H(2)O, NH(3), OH(-), F(-), Cl(-), Br(-), H(2)S, CH(3)S(-), SCN(-), CN(-), PH(3), CO, CH(3)(-), H(-), C(2)H(4)). The trans effect is explained in terms of sigma-donation and pi-back-donation whose relative strengths are quantified by the changes of electron occupations of 5d platinum atomic orbitals. The sigma-donation strength is linearly correlated with the Pt-H(2)O (leaving ligand) bond length (trans influence). The kinetic trans effect strength correlates proportionally with the sigma-donation ability of the trans-ligand except the ligands with strong pi-back-donation ability that stabilizes transition state structure. The sigma-donation ability of the ligand is dependent on the sigma-donation strength of the ligand in the trans position. Therefore the trans effect caused by sigma-donation can be understood as a competition between the trans-ligands for the opportunity to donate electron density to the central Pt(II) atom. The influence of the trans effect on the reaction mechanism is also shown. For ligands with a very strong sigma-donation (e.g. CH(3)(-) and H(-)), the substitution proceeds by a dissociative interchange (I(d)) mechanism. Ligands with strong pi-back donation ability (e.g. C(2)H(4)) stabilize the pentacoordinated intermediate and the substitution proceeds by a two step associative mechanism. For ligands with weak sigma-donation and pi-back-donation abilities, the highest activation barriers have to be overcome and substitutions can be described by an associative interchange (I(a)) mechanism. The results are supported by the energy decomposition and the natural orbital analysis.     

Reaction of cytochrome P450BM3 and peroxynitrite yields nitrosyl complex

Peroxynitrite has come into the spotlight in recent years. Its effects on proteins have been implicated in several diseases such as acute lung injury, rheumatoid arthritis, implant rejection, artherosclerosis, Parkinson's disease, and Alzheimer's disease. Peroxynitrite is thought to inactivate a variety of proteins including thiolate-ligated heme proteins such as cytochrome P450 2B1 and PGI2 synthase, through the nitration of tyrosine residues. In previous studies it was reported that thiolate-ligated heme enzymes react with peroxynitrite to form a ferryl intermediate. In an effort to spectroscopically characterize this species in P450BM3, we discovered that the peroxynitrite-generated intermediate is not an FeIVoxo, but rather an iron-nitrosyl [FeNO]6 complex. We present density functional calculations as well as M?ssbauer and stopped-flow spectroscopic characterizations of the peroxynitrite-generated intermediate in P450BM3.     

H2O2-mediated oxidation of zero-valent silver and resultant interactions among silver nanoparticles, silver ions, and reactive oxygen species

The H(2)O(2)-mediated oxidation of silver nanoparticles (AgNPs) over a range of pH (3.0-14.0) is investigated here, and an electron charging-discharging model capable of describing the experimental results obtained is developed. AgNPs initially react with H(2)O(2) to form Ag(+) and superoxide, with these products subsequently reacting to reform AgNPs (in-situ-formed AgNPs) via an electron charging-discharging mechanism. Our experimental results show that the AgNP reactivity toward H(2)O(2) varies significantly with pH, with the variation at high pH (>10) due particularly to the differences in the reactivity of H(2)O(2) and its conjugate base HO(2)(-) with AgNPs whereas at lower pH (3-10) the pH dependence of H(2)O(2) decay is accounted for, at least in part, by the pH dependence of the rate of superoxide disproportionation. Our results further demonstrate that the in-situ-formed AgNPs resulting from the superoxide-mediated reduction of Ag(+) have a different size and reactivity compared to those of the citrate-stabilized particles initially present. The turnover frequency for AgNPs varies significantly with pH and is as high as 1776.0 min(-1) at pH 11.0, reducing to 144.2 min(-1) at pH 10.0 and 3.2 min(-1) at pH 3.0.     

NO and O2 reactivities of synthetic functional models of nitric oxide reductase and cytochrome c oxidase

Though nitric oxide reductase (NOR) and cytochrome c oxidase (CcO) have similar active sites, they exhibit quite different functions. While NOR reduces NO to N(2)O, CcO reduces O(2) to H(2)O. Further, CcO is reversibly inhibited by NO, the substrate for NOR, and NOR is reversibly inhibited by O(2), which is the substrate for CcO. Over the past decade several structural and functional models of these enzymes have been reported. The mimics have been used to understand the reaction mechanism of these enzymes and develop structure function correlations for these active sites. This article summarizes these recent developments with particular stress on the reactivities of functional mimics of CcO with NO and functional mimics of NOR with O(2).