Electronic structure of high-spin iron(III)-alkylperoxo complexes and its relation to low-spin analogues: reaction coordinate of O-O bond homolysis.

The spectroscopic properties of the high-spin Fe(III)-alkylperoxo model complex [Fe(6-Me(3)TPA)(OH(x))(OO(t)Bu)](x)(+) (1; TPA = tris(2-pyridylmethyl)amine, (t)Bu = tert-butyl, x = 1 or 2) are defined and related to density functional calculations of corresponding models in order to determine the electronic structure and reactivity of this system. The Raman spectra of 1 show four peaks at 876, 842, 637, and 469 cm(-1) that are assigned with the help of normal coordinate analysis, and corresponding force constants have been determined to be 3.55 mdyn/A for the O-O and 2.87 mdyn/A for the Fe-O bond. Complex 1 has a broad absorption feature around 560 nm that is assigned to a charge-transfer (CT) transition from the alkylperoxo to a t(2g) d orbital of Fe(III) with the help of resonance Raman profiles and MCD spectroscopy. An additional contribution to the Fe-O bond arises from a sigma interaction between and an e(g) d orbital of iron. The electronic structure of 1 is compared to the related low-spin model complex [Fe(TPA)(OH(x))(OO(t)Bu)](x)(+) and the reaction coordinate for O-O homolysis is explored for both the low-spin and the high-spin Fe(III)-alkylperoxo systems. Importantly, there is a barrier for homolytic cleavage of the O-O bond on the high-spin potential energy surface that is not present for the low-spin complex, which is therefore nicely set up for O-O homolysis. This is reflected by the electronic structure of the low-spin complex having a strong Fe-O and a weak O-O bond due to a strong Fe-O sigma interaction. In addition, the reaction coordinate of the Fe-O homolysis has been investigated, which is a possible decay pathway for the high-spin system, but which is thermodynamically unfavorable for the low-spin complex.     

Electronic structure and reactivity of low-spin Fe(III)-hydroperoxo complexes: comparison to activated bleomycin

The spectroscopic properties, electronic structure, and reactivity of the low-spin Fe(III)-hydroperoxo complex [Fe(N4Py)(OOH)](2+) (1, N4Py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) are investigated in comparison to those of activated bleomycin (ABLM). Complex 1 is characterized by Raman features at 632 (Fe-O stretch) and 790 cm(-1) (O-O stretch), corresponding to a strong Fe-O bond (force constant 3.62 mdyn/A) and a weak O-O bond (3.05 mdyn/A). The UV-vis spectrum of 1 shows a broad absorption band around 550 nm that is assigned to a charge-transfer transition from the hydroperoxo to a t(2g) d orbital of Fe(III) using resonance Raman and MCD spectroscopies and density functional (DFT) calculations. Compared to low-spin [Fe(TPA)(OH(x))(OO(t)Bu)](x+)(TPA = tris(2-pyridylmethyl)amine, x = 1 or 2), an overall similar Fe-OOR bonding results for low-spin Fe(III)-alkylperoxo and -hydroperoxo species. Correspondingly, both systems show similar reactivities and undergo homolytic cleavage of the O-O bond. From the DFT calculations, this reaction is more endothermic for 1 due to the reduced stabilization of the .OH radical compared to .O(t)Bu and the absence of the hydroxo ligand that helps to stabilize the resulting Fe(IV)=O species. In contrast, ABLM has a somewhat different electronic structure where no pi donor bond between the hydroperoxo ligand and iron(III) is present [Neese, F.; Zaleski, J. M.; Loeb-Zaleski, K.; Solomon, E. I. J. Am. Chem. Soc. 2000, 122, 11703]. Possible reaction pathways for ABLM are discussed in relation to known experimental results.     

Electronic structure and reactivity of high-spin iron--alkyl- and--pterinperoxo complexes

The spectroscopic properties and electronic structure of the four-coordinate high-spin [FeIII(L3)(OOtBu)]+ complex (1; L3 = hydrotris(3-tert-butyl-5-isopropyl-1-pyrazolyl)borate; tBu = tert-butyl) are investigated and compared to the six-coordinated high-spin [Fe(6-Me3TPA)(OHx)(OOtBu)]x+ system (TPA = tris(2-pyridylmethyl)amine, x = 1 or 2) studied earlier [Lehnert, N.; Ho, R. Y. N.; Que, L., Jr.; Solomon, E. I. J. Am. Chem. Soc. 2001, 123, 12802-12816]. Complex 1 is characterized by Raman features at 889 and 830 cm-1 which are assigned to the O-O stretch (mixed with the symmetric C-C stretch) and a band at 625 cm-1 that corresponds to nu(Fe-O). The UV-vis spectrum shows a charge-transfer (CT) transition at 510 nm from the alkylperoxo pi v* (v = vertical to C-O-O plane) to a d orbital of Fe(III). A second CT is identified from MCD at 370 nm that is assigned to a transition from pi h* (h = horizontal to C-O-O plane) to an Fe(III) d orbital. For the TPA complex the pi v* CT is at 560 nm while the pi h* CT is to higher energy than 250 nm. These spectroscopic differences between four- and six-coordinate Fe(III)-OOR complexes are interpreted on the basis of their different ligand fields. In addition, the electronic structure of Fe-OOPtn complexes with the biologically relevant pterinperoxo ligand are investigated. Substitution of the tert-butyl group in 1 by pterin leads to the corresponding Fe(III)-OOPtn species (2), which shows a stronger electron donation from the peroxide to Fe(III) than 1. This is related to the lower ionization potential of pterin. Reduction of 2 by one electron leads to the Fe(II)-OOPtn complex (3), which is relevant as a model for potential intermediates in pterin-dependent hydroxylases. However, in the four-coordinate ligand field of 3, the additional electron is located in a nonbonding d orbital of iron. Hence, the pterinperoxo ligand is not activated for heterolytic cleavage of the O-O bond in this system. This is also evident from the calculated reaction energies that are endothermic by at least 20 kcal/mol.     

Rational tuning of the thiolate donor in model complexes of superoxide reductase: direct evidence for a trans influence in Fe(III)-OOR complexes

Iron peroxide species have been identified as important intermediates in a number of nonheme iron as well as heme-containing enzymes, yet there are only a few examples of such species either synthetic or biological that have been well characterized. We describe the synthesis and structural characterization of a new series of five-coordinate (N4S(thiolate))Fe(II) complexes that react with tert-butyl hydroperoxide ((t)BuOOH) or cumenyl hydroperoxide (CmOOH) to give metastable alkylperoxo-iron(III) species (N4S(thiolate)Fe(III)-OOR) at low temperature. These complexes were designed specifically to mimic the nonheme iron active site of superoxide reductase, which contains a five-coordinate iron(II) center bound by one Cys and four His residues in the active form of the protein. The structures of the Fe(II) complexes are analyzed by X-ray crystallography, and their electrochemical properties are assessed by cyclic voltammetry. For the Fe(III)-OOR species, low-temperature UV-vis spectra reveal intense peaks between 500-550 nm that are typical of peroxide to iron(III) ligand-to-metal charge-transfer (LMCT) transitions, and EPR spectroscopy shows that these alkylperoxo species are all low-spin iron(III) complexes. Identification of the vibrational modes of the Fe(III)-OOR unit comes from resonance Raman (RR) spectroscopy, which shows nu(Fe-O) modes between 600-635 cm(-1) and nu(O-O) bands near 800 cm(-1). These Fe-O stretching frequencies are significantly lower than those found in other low-spin Fe(III)-OOR complexes. Trends in the data conclusively show that this weakening of the Fe-O bond arises from a trans influence of the thiolate donor, and density functional theory (DFT) calculations support these findings. These results suggest a role for the cysteine ligand in SOR, and are discussed in light of the recent assessments of the function of the cysteine ligand in this enzyme.     

Kinetic analysis of the conversion of nonheme (alkylperoxo)iron(III) species to iron(IV) complexes

Low-spin mononuclear (alkylperoxo)iron(III) complexes decompose by peroxide O-O bond homolysis to form iron(IV) species. We examined the kinetics of previously reported homolysis reactions for (alkylperoxo)iron(III) intermediates supported by TPA (tris(2-pyridylmethyl)amine) in CH3CN solution and promoted by pyridine N-oxide, and by BPMCN (N,N-bis(2-pyridylmethyl)-N,N-dimethyl-trans-1,2-diaminocyclohexane) in its cis-beta configuration in CH3CN and CH2Cl2, as well as for the previously unreported chemistry of TPA and 5-Me3TPA intermediates in acetone. Each of these reactions forms an oxoiron(IV) complex, except for the beta-BPMCN reaction in CH2Cl2 that yields a novel (hydroxo)(alkylperoxo)iron(IV) product. Temperature-dependent rate measurements suggest a common reaction trajectory for each of these reactions and verify previous theoretical estimates of a ca. 60 kJ/mol enthalpic barrier to homolysis. However, both the tetradentate supporting ligand and exogenous ligands in the sixth octahedral coordination site significantly perturb the homolyses, such that observed rates can vary over 2 orders of magnitude at a given temperature. This is manifested as a compensation effect in which increasing activation enthalpy is offset by increasingly favorable activation entropy. Moreover, the applied kinetic model is consistent with geometric isomerism in the low-spin (alkylperoxo)iron(III) intermediates, wherein the alkylperoxo ligand is coordinated in either of the inequivalent cis sites afforded by the nonheme ligands.     

Spectroscopic study of [Fe2O2(5-Et3-TPA)2]3+: nature of the Fe2O2 diamond core and its possible relevance to high-valent binuclear non-heme enzyme intermediates

The spectroscopic properties and electronic structure of an Fe(2)(III,IV) bis-mu-oxo complex, [Fe(2)O(2)(5-Et(3)-TPA)(2)](ClO(4))(3) where 5-Et(3)-TPA = tris(5-ethyl-2-pyridylmethyl)amine, are explored to determine the molecular origins of the unique electronic and geometric features of the Fe(2)O(2) diamond core. Low-temperature magnetic circular dichroism (MCD) allows the two features in the broad absorption envelope (4000-30000 cm(-)(1)) to be resolved into 13 transitions. Their C/D ratios and transition polarizations from variable temperature-variable field MCD saturation behavior indicate that these divide into three types of electronic transitions; t(2) --> t(2) involving excitations between metal-based orbitals with pi Fe-O overlap (4000-10000 cm(-)(1)), t(2)/t(2) --> e involving excitations to metal-based orbitals with sigma Fe-O overlap (12500-17000 cm(-)(1)) and LMCT (17000-30000 cm(-)(1)) and allows transition assignments and calibration of density functional calculations. Resonance Raman profiles show the C(2)(h)() geometric distortion of the Fe(2)O(2) core results in different stretching force constants for adjacent Fe-O bonds (k(str)(Fe-O(long)) = 1.66 and k(str)(Fe-O(short)) = 2.72 mdyn/A) and a small ( approximately 20%) difference in bond strength between adjacent Fe-O bonds. The three singly occupied pi-metal-based orbitals form strong superexchange pathways which lead to the valence delocalization and the S = (3)/(2) ground state. These orbitals are key to the observed reactivity of this complex as they overlap with the substrate C-H bonding orbital in the best trajectory for hydrogen atom abstraction. The electronic structure implications of these results for the high-valent enzyme intermediates X and Q are discussed.     

Characterization of a high-spin non-heme Fe(III)-OOH intermediate and its quantitative conversion to an Fe(IV)═O complex

We have generated a high-spin Fe(III)-OOH complex supported by tetramethylcyclam via protonation of its conjugate base and characterized it in detail using various spectroscopic methods. This Fe(III)-OOH species can be converted quantitatively to an Fe(IV)═O complex via O-O bond cleavage; this is the first example of such a conversion. This conversion is promoted by two factors: the strong Fe(III)-OOH bond, which inhibits Fe-O bond lysis, and the addition of protons, which facilitates O-O bond cleavage. This example provides a synthetic precedent for how O-O bond cleavage of high-spin Fe(III)-peroxo intermediates of non-heme iron enzymes may be promoted.     

A density functional study of O-O bond cleavage for a biomimetic non-heme iron complex demonstrating an Fe(v)-intermediate

Density functional theory using the B3LYP hybrid functional has been employed to investigate the reactivity of Fe(TPA) complexes (TPA = tris(2-pyridylmethyl)amine), which are known to catalyze stereospecific hydrocarbon oxidation when H(2)O(2) is used as oxidant. The reaction pathway leading to O-O bond heterolysis in the active catalytic species Fe(III)(TPA)-OOH has been explored, and it is shown that a high-valent iron-oxo intermediate is formed, where an Fe(V) oxidation state is attained, in agreement with previous suggestions based on experiments. In contrast to the analogous intermediate [(Por.)Fe(IV)=O](+1) in P450, the TPA ligand is not oxidized, and the electrons are extracted almost exclusively from the mononuclear iron center. The corresponding homolytic O-O bond cleavage, yielding the two oxidants Fe(IV)=O and the OH. radical, has also been considered, and it is shown that this pathway is inaccessible in the hydrocarbon oxidation reaction with Fe(TPA) and hydrogen peroxide. Investigations have also been performed for the O-O cleavage in the Fe(III)(TPA)-alkylperoxide species. In this case, the barrier for O-O homolysis is found to be slightly lower, leading to loss of stereospecificity and supporting the experimental conclusion that this is the preferred pathway for alkylperoxide oxidants. The difference between hydroperoxide and alkylperoxide as oxidant derives from the higher O-O bond strength for hydrogen peroxide (by 8.0 kcal/mol).     

Nature of the Fe-O2 bonding in oxy-myoglobin: effect of the protein

The nature of the Fe-O2 bonding in oxy-myoglobin was probed by theoretical calculations: (a) QM/MM (hybrid quantum mechanical/molecular mechanical) calculations using DFT/MM and CASSCF/MM methods and (b) gas-phase calculations using DFT (density functional theory) and CASSCF (complete active space self-consistent field) methods. Within the protein, the O2 is hydrogen bonded by His64 and the complex feels the bulk polarity of the protein. Removal of the protein causes major changes in the complex. Thus, while CASSCF/MM and DFT/MM are similar in terms of state constitution, degree of O2 charge, and nature of the lowest triplet state, the gas-phase CASSCF(g) species is very different. Valence bond (VB) analysis of the CASSCF/MM wave function unequivocally supports the Weiss bonding mechanism. This bonding arises by electron transfer from heme-Fe(II) to O2 and the so formed species coupled then to a singlet state Fe(III)-O2(-) that possesses a dative sigma(Fe-O) bond and a weakly coupled pi(Fe-O2) bond pair. The bonding mechanism in the gas phase is similar, but now the sigma(Fe-O) bond involves higher back-donation from O2(-) to Fe(III), while the constituents of pi(Fe-O2) bond pair have greater delocalization tails. The protein thus strengthens the Fe(III)-O2(-) character of the complex and thereby affects its bonding features and the oxygen binding affinity of Mb. The VB model is generalized, showing how the protein or the axial ligand of the oxyheme complex can determine the nature of its bonding in terms of the blend of the three bonding models: Weiss, Pauling, and McClure-Goddard.     

Spectroscopic properties and electronic structure of five- and six-coordinate iron(II) porphyrin NO complexes: Effect of the axial N-donor ligand

In this paper, the differences in the spectroscopic properties and electronic structures of five- and six-coordinate iron(II) porphyrin NO complexes are explored using [Fe(TPP)(NO)] (1; TPP = tetraphenylporphyrin) and [Fe(TPP)(MI)(NO)] (2; MI = 1-methylimidazole) type systems. Binding of N-donor ligands in axial position trans to NO to five-coordinate complexes of type 1 is investigated using UV-vis absorption and 1H NMR spectroscopies. This way, the corresponding binding constants Keq are determined and the 1H NMR spectra of 1 and 2 are assigned for the first time. In addition, 1H NMR allows for the determination of the degree of denitrosylation in solutions of 1 with excess base. The influence of the axial ligand on the properties of the coordinated NO is then investigated. Vibrational spectra (IR and Raman) of 1 and 2 are presented and assigned using isotope substitution and normal-coordinate analysis. Obtained force constants are 12.53 (N-O) and 2.98 mdyn/A (Fe-NO) for 1 compared to 11.55 (N-O) and 2.55 mdyn/A (Fe-NO) for 2. Together with the NMR results, this provides experimental evidence that binding of the trans ligand weakens the Fe-NO bond. The principal bonding schemes of 1 and 2 are very similar. In both cases, the Fe-N-O subunit is strongly bent. Donation from the singly occupied pi* orbital of NO into d(z2) of iron(II) leads to the formation of an Fe-NO sigma bond. In addition, a medium-strong pi back-bond is present in these complexes. The most important difference in the electronic structures of 1 and 2 occurs for the Fe-NO sigma bond, which is distinctively stronger for 1 in agreement with the experimental force constants. The increased sigma donation from NO in 1 also leads to a significant transfer of spin density from NO to iron, as has been shown by magnetic circular dichroism (MCD) spectroscopy in a preceding Communication (Praneeth, V. K. K.; Neese, F.; Lehnert, N. Inorg. Chem. 2005, 44, 2570-2572). This is confirmed by the 1H NMR results presented here. Hence, further experimental and computational evidence is provided that complex 1 has noticeable Fe(I)NO+ character relative to 2, which is an Fe(II)NO(radical) complex. Finally, using MCD theory and quantum chemical calculations, the absorption and MCD C-term spectra of 1 and 2 are assigned for the first time.     

End-on and side-on peroxo derivatives of non-heme iron complexes with pentadentate ligands: models for putative intermediates in biological iron/dioxygen chemistry

Mononuclear iron(III) species with end-on and side-on peroxide have been proposed or identified in the catalytic cycles of the antitumor drug bleomycin and a variety of enzymes, such as cytochrome P450 and Rieske dioxygenases. Only recently have biomimetic analogues of such reactive species been generated and characterized at low temperatures. We report the synthesis and characterization of a series of iron(II) complexes with pentadentate N5 ligands that react with H(2)O(2) to generate transient low-spin Fe(III)-OOH intermediates. These intermediates have low-spin iron(III) centers exhibiting hydroperoxo-to-iron(III) charge-transfer bands in the 500-600-nm region. Their resonance Raman frequencies, nu(O)(-)(O), near 800 cm(-)(1) are significantly lower than those observed for high-spin counterparts. The hydroperoxo-to-iron(III) charge-transfer transition blue-shifts and the nu(O)(-)(O) of the Fe-OOH unit decreases as the N5 ligand becomes more electron donating. Thus, increasing electron density at the low-spin Fe(III) center weakens the O-O bond, in accord with conclusions drawn from published DFT calculations. The parent [(N4Py)Fe(III)(eta(1)-OOH)](2+) (1a) ion in this series (N4Py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) can be converted to its conjugate base, which is demonstrated to be a high-spin iron(III) complex with a side-on peroxo ligand, [(N4Py)Fe(III)(eta(2)-O(2))](+) (1b). A detailed analysis of 1a and 1b by EPR and M?ssbauer spectroscopy provides insights into their electronic properties. The orientation of the observed (57)Fe A-tensor of 1a can be explained with the frequently employed Griffith model provided the rhombic component of the ligand field, determined by the disposition of the hydroperoxo ligand, is 45 degrees rotated relative to the octahedral field. EXAFS studies of 1a and 1b reveal the first metrical details of the iron-peroxo units in this family of complexes: [(N4Py)Fe(III)(eta(1)-OOH)](2+) has an Fe-O bond of 1.76 A, while [(N4Py)Fe(III)(eta(2)-O(2))](+) has two Fe-O bonds of 1.93 A, values which are in very good agreement with results obtained from DFT calculations.     

Spin density distribution in five- and six-coordinate iron(II)-porphyrin NO complexes evidenced by magnetic circular dichroism spectroscopy

Using magnetic circular dichroism (MCD) spectroscopy together with DFT calculations, the spin density distributions in five-coordinate [Fe(TPP)(NO)] (I) and six-coordinate [Fe(TPP)(MI)(NO)] (II, MI = 1-methylimidazole) are defined. In the five-coordinate complex, a strong Fe-NO sigma bond between pi(*)(h) and d(z)(2) is present that leads to a large transfer of spin density from the NO ligand to Fe(II) corresponding to an electronic structure with noticeable Fe(I)-NO(+) character. Consequently, the MCD spectrum is dominated by paramagnetic C-term contributions. On coordination of the sixth ligand, the spin density is pushed back from the iron toward the NO ligand, resulting in an Fe(II)-NO(radical) type of electronic structure. This is reflected by the fact that the MCD spectrum is dominated by diamagnetic contributions.     

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.     

Theoretical study of the mechanism of oxoiron(IV) formation from H2O2 and a nonheme iron(II) complex: O-O cleavage involving proton-coupled electron transfer

It has recently been shown that the nonheme oxoiron(IV) species supported by the 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane ligand (TMC) can be generated in near-quantitative yield by reacting [Fe(II)(TMC)(OTf)(2)] with a stoichiometric amount of H(2)O(2) in CH(3)CN in the presence of 2,6-lutidine (Li, F.; England, J.; Que, L., Jr. J. Am. Chem. Soc. 2010, 132, 2134-2135). This finding has major implications for O-O bond cleavage events in both Fenton chemistry and nonheme iron enzymes. To understand the mechanism of this process, especially the intimate details of the O-O bond cleavage step, a series of density functional theory (DFT) calculations and analyses have been carried out. Two distinct reaction paths (A and B) were identified. Path A consists of two principal steps: (1) coordination of H(2)O(2) to Fe(II) and (2) a combination of partial homolytic O-O bond cleavage and proton-coupled electron transfer (PCET). The latter combination renders the rate-limiting O-O cleavage effectively a heterolytic process. Path B proceeds via a simultaneous homolytic O-O bond cleavage of H(2)O(2) and Fe-O bond formation. This is followed by H abstraction from the resultant Fe(III)-OH species by an ?OH radical. Calculations suggest that path B is plausible in the absence of base. However, once 2,6-lutidine is added to the reacting system, the reaction barrier is lowered and more importantly the mechanistic path switches to path A, where 2,6-lutidine plays an essential role as an acid-base catalyst in a manner similar to how the distal histidine or glutamate residue assists in compound I formation in heme peroxidases. The reaction was found to proceed predominantly on the quintet spin state surface, and a transition to the triplet state, the experimentally known ground state for the TMC-oxoiron(IV) species, occurs in the last stage of the oxoiron(IV) formation process.     

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.     

The reaction of [Fe(pic)3] with hydrogen peroxide: a UV-visible and EPR spectroscopic study (Hpic = picolinic acid)

The Gif family of catalysts, based on an iron salt and O2 or H2O2 in pyridine, allows the oxygenation of cyclic saturated hydrocarbons to ketones and alcohols under mild conditions. The reaction between [Fe(pic)3] and hydrogen peroxide in pyridine under GoAgg(III)(Fe(III)/Hpic catalyst) conditions was investigated by UV-visible spectrophotometry. Reactions were monitored at 430 and 520 nm over periods ranging from a few minutes to several hours at 20 degrees C. A number of kinetically stable intermediates were detected, and their relevance to the processes involved in the assembly of the active GoAgg(III) catalyst was determined by measuring the kinetics in the presence and absence of cyclohexane. EPR measurements at 110 K using hydrogen peroxide and t-BuOOH as oxidants were used to further probe these intermediates. Our results indicate that in wet pyridine [Fe(pic)3] undergoes reversible dissociation of one picolinate ligand, establishing an equilibrium with [Fe(pic)2(py)(OH)]. Addition of aqueous hydrogen peroxide rapidly generates the high-spin complex [Fe(pic)2(py)(eta1-OOH)] from the labilised hydroxy species. Subsequently the hydroperoxy species undergoes homolysis of the Fe-O bond, generating HOO. and [Fe(pic)2(py)2], the active oxygenation catalyst.     

Spectroscopic and computational studies of Co2+corrinoids: spectral and electronic properties of the biologically relevant base-on and base-off forms of Co2+cobalamin

Co(2+)cobalmain (Co(2+)Cbl) is implicated in the catalytic cycles of all adenosylcobalamin (AdoCbl)-dependent enzymes, as in each case catalysis is initiated through homolytic cleavage of the cofactor's Co-C bond. The rate of Co-C bond homolysis, while slow for the free cofactor, is accelerated by 12 orders of magnitude when AdoCbl is bound to the protein active site, possibly through enzyme-mediated stabilization of the post-homolysis products. As an essential step toward the elucidation of the mechanism of enzymatic Co-C bond activation, we employed electronic absorption (Abs), magnetic circular dichroism (MCD), and resonance Raman spectroscopies to characterize the electronic excited states of Co(2+)Cbl and Co(2+)cobinamide (Co(2+)Cbi(+), a cobalamin derivative that lacks the nucleotide loop and 5,6-dimethylbenzimazole (DMB) base and instead binds a water molecule in the lower axial position). Although relatively modest differences exist between the Abs spectra of these two Co(2+)corrinoid species, MCD data reveal that substitution of the lower axial ligand gives rise to dramatic changes in the low-energy region where Co(2+)-centered ligand field transitions are expected to occur. Our quantitative analysis of these spectral changes within the framework of time-dependent density functional theory (TD-DFT) calculations indicates that corrin-based pi --> pi transitions, which dominate the Co(2+)corrinoid Abs spectra, are essentially insulated from perturbations of the lower ligand environment. Contrastingly, the Co(2+)-centered ligand field transitions, which are observed here for the first time using MCD spectroscopy, are extremely sensitive to alterations in the Co(2+) ligand environment and thus may serve as excellent reporters of enzyme-induced perturbations of the Co(2+) state. The power of this combined spectroscopic/computational methodology for studying Co(2+)corrinoid/enzyme active site interactions is demonstrated by the dramatic changes in the MCD spectrum as Co(2+)Cbi(+) binds to the adenosyltransferase CobA.     

Synthetic models for the cysteinate-ligated non-heme iron enzyme superoxide reductase: observation and structural characterization by XAS of an Fe(III)-OOH intermediate

Superoxide reductases (SORs) belong to a new class of metalloenzymes that degrade superoxide by reducing it to hydrogen peroxide. These enzymes contain a catalytic iron site that cycles between the Fe(II) and Fe(III) states during catalysis. A key step in the reduction of superoxide has been suggested to involve HO(2) binding to Fe(II), followed by innersphere electron transfer to afford an Fe(III)-OO(H) intermediate. In this paper, the mechanism of the superoxide-induced oxidation of a synthetic ferrous SOR model ([Fe(II)(S(Me2)N(4)(tren))](+) (1)) to afford [Fe(III)(S(Me2)N(4)(tren)(solv))](2+) (2-solv) is reported. The XANES spectrum shows that 1 remains five-coordinate in methanolic solution. Upon reaction of 1 with KO(2) in MeOH at -90 degrees C, an intermediate (3) is formed, which is characterized by a LMCT band centered at 452(2780) nm, and a low-spin state (S = 1/2), based on its axial EPR spectrum (g(perpendicular) = 2.14; g(parallel) = 1.97). Hydrogen peroxide is detected in this reaction, using both (1)H NMR spectroscopy and a catalase assay. Intermediate 3 is photolabile, so, in lieu of a Raman spectrum, IR was used to obtain vibrational data for 3. At low temperatures, a nu(O-O) Fermi doublet is observed in the IR at 788(2) and 781(2) cm(-)(1), which collapses into a single peak at 784 cm(-1) upon the addition of D(2)O. This vibrational peak diminishes in intensity over time and essentially disappears after 140 s. When 3 is generated using an (18)O-labeled isotopic mixture of K(18)O(2)/K(16)O(2) (23.28%), the vibration centered at 784 cm(-1) shifts to 753 cm(-1). This new vibrational peak is close to that predicted (740 cm(-1)) for a diatomic (18)O-(18)O stretch. In addition, a nu(O-O) vibrational peak assigned to free hydrogen peroxide is also observed (nu(O-O) = 854 cm(-1)) throughout the course of the reaction between Fe(II)-1 and superoxide and is strongest after 100 s. XAS studies indicate that 3 possesses one sulfur scatterer at 2.33(2) A and four nitrogen scatterers at 2.01(1) A. Addition of two Fe-O shells, each containing one oxygen, one at 1.86(3) A and one at 2.78(3) A, improved the EXAFS fits, suggesting that 3 is an end-on peroxo or hydroperoxo complex, [Fe(III)(S(Me2)N(4)(tren))(OO(H))](+). Upon warming above -50 degrees C, 3 is converted to 2-MeOH. In methanol and methanol:THF (THF = tetrahydrofuran) solvent mixtures, 2-MeOH is characterized by a LMCT band at lambda(max) = 511(1765) nm, an intermediate spin-state (S = 3/2), and, on the basis of EXAFS, a relatively short Fe-O bond (assigned to a coordinated methanol or methoxide) at 1.94(10) A. Kinetic measurements in 9:1 THF:MeOH at 25 degrees C indicate that 3 is formed near the diffusion limit upon addition of HO(2) to 1 and converts to 2-MeOH at a rate of 65(1) s(-1), which is consistent with kinetic studies involving superoxide oxidation of the SOR iron site.     

Reactivity of mononuclear alkylperoxo copper(II) complex. O-O bond cleavage and C-H bond activation

A detailed reactivity study has been carried out for the first time on a new mononuclear alkylperoxo copper(II) complex, which is generated by the reaction of copper(II) complex supported by the bis(pyridylmethyl)amine tridentate ligand containing a phenyl group at the 6-position of the pyridine donor groups and cumene hydroperoxide (CmOOH) in CH3CN. The cumylperoxo copper(II) complex thus obtained has been found to undergo homolytic cleavage of the O-O bond and induce C-H bond activation of exogenous substrates, providing important insights into the catalytic mechanism of copper monooxygenases.