The role of the sulfonium linkage in the stabilization of the ferrous form of myeloperoxidase: a comparison with lactoperoxidase

In all mammalian peroxidases, the heme is covalently attached to the protein via two ester linkages between conserved aspartate (Asp94) and glutamate residues (Glu242) and modified methyl groups on pyrrole rings A and C. Only myeloperoxidase has an additional sulfonium ion linkage between the sulfur atom of the conserved methionine 243 and the beta-carbon of the vinyl group on pyrrole ring A. Upon reduction from Fe(III) to Fe(II), lactoperoxidase (LPO) but not myeloperoxidase (MPO) is shown to adopt three distinct active site conformations which depend on pH and time. Comparative spectroscopic analysis (UV-Vis absorption and resonance Raman) of the ferrous forms of LPO, wild-type MPO and the variants Asp94Val, Glu242Gln, Met243Thr and Met243Val clearly demonstrate that a single, stable ferrous form of MPO is present only in those proteins which retain an intact sulfonium linkage. By contrast, both ferrous Met243Thr and Met243Val can assume two conformations. They resemble ferrous LPO, being five-coordinated high-spin species that are distinguished by the strength of the proximal Fe-histidine bond. This bond weakens with time or decreasing pH, as indicated by the Fe-histidine stretching bands.     

Role of the covalent glutamic acid 242-heme linkage in the formation and reactivity of redox intermediates of human myeloperoxidase

In human myeloperoxidase the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu242 and Asp94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met243 and the beta-carbon of the vinyl group on pyrrole ring A. In the present study, wild-type recombinant myeloperoxidase (recMPO) and the variant Glu242Gln were produced in Chinese hamster ovary cells and investigated in a comparative sequential-mixing stopped-flow study in order to elucidate the role of the Glu242-heme ester linkage in the individual reaction steps of both the halogenation and peroxidase cycle. Disruption of the ester bond increased heme flexibility, blue shifted the UV-vis spectrum, and, compared with recMPO, decelerated cyanide binding (1.25 x 10(4) versus 1.6 x 10(6) M(-)(1) s(-)(1) at pH 7 and 25 degrees C) as well as compound I formation mediated by either hydrogen peroxide (7.8 x 10(5) versus 1.9 x 10(7) M(-)(1) s(-)(1)) or hypochlorous acid (7.5 x 10(5) versus 2.3 x 10(7) M(-)(1) s(-)(1)). The overall chlorination and bromination activity of Glu242Gln was 2.0% and 24% of recMPO. The apparent bimolecular rate constants of compound I reduction by chloride (65 M(-)(1) s(-)(1)), bromide (5.4 x 10(4) M(-)(1) s(-)(1)), iodide (6.4 x 10(5) M(-)(1) s(-)(1)), and thiocyanate (2.2 x10(5) M(-)(1) s(-)(1)) were 500, 25, 21, and 63 times decreased compared with recMPO. By contrast, Glu242Gln compound I reduction by tyrosine was only 5.4 times decreased, whereas tyrosine-mediated compound II reduction was 60 times slower compared with recMPO. The effects of exchange of Glu242 on electron transfer reactions are discussed.     

The sulfonium ion linkage in myeloperoxidase. Direct spectroscopic detection by isotopic labeling and effect of mutation.

The heme group of myeloperoxidase is covalently linked via two ester bonds to the protein and a unique sulfonium ion linkage involving Met(243). Mutation of Met(243) into Thr, Gln, and Val, which are the corresponding residues of eosinophil peroxidase, lactoperoxidase, and thyroid peroxidase, respectively, and into Cys was performed. The Soret band in the optical absorbance spectrum in the oxidized mutants is now found at approximately 411 nm. Both the pyridine hemochrome spectra and resonance Raman spectra of the mutants are affected by the mutation. In the Met(243) mutants the affinity for chloride has decreased 100-fold. All mutants have lost their chlorination activity, except for the M243T mutant, which still has 15% activity left. By Fourier transform infared difference spectroscopy it was possible to specifically detect the (13)CD(3)-labeled methionyl sulfonium ion linkage. We conclude that the sulfonium ion linkage serves two roles. First, it serves as an electron-withdrawing substituent via its positive charge, and, second, together with its neighboring residue Glu(242), it appears to be responsible for the lower symmetry of the heme group and distortion from the planar conformation normally seen in heme-containing proteins.     

X-ray crystal structure and characterization of halide-binding sites of human myeloperoxidase at 1.8 A resolution

The x-ray crystal structure of human myeloperoxidase has been extended to 1.8 A resolution, using x-ray data recorded at -180 degrees C (r = 0.197, free r = 0.239). Results confirm that the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu(242) and Asp(94) and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met(243) and the beta-carbon of the vinyl group on pyrrole ring A. In the native enzyme a bound chloride ion has been identified at the amino terminus of the helix containing the proximal His(336). Determination of the x-ray crystal structure of a myeloperoxidase-bromide complex (r = 0.243, free r = 0.296) has shown that this chloride ion can be replaced by bromide. Bromide is also seen to bind, at partial occupancy, in the distal heme cavity, in close proximity to the distal His(95), where it replaces the water molecule hydrogen bonded to Gln(91). The bromide-binding site in the distal cavity appears to be the halide-binding site responsible for shifts in the Soret band of the absorption spectrum of myeloperoxidase. It is proposed that halide binding to this site inhibits the enzyme by effectively competing with H(2)O(2) for access to the distal histidine, whereas in compound I, the same site may be the halide substrate-binding site.     

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

 The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. Received: 17 July 1996 / Accepted: 28 November 1996     

Influence of the covalent heme-protein bonds on the redox thermodynamics of human myeloperoxidase

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its (electronic) structure and biophysical and chemical properties. Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants with disrupted heme-protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. It turns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positive reduction potential (E°' = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. By contrast, upon disruption of the MPO-typical sulfonium ion linkage, the reduction potential is significantly lower (-0.182 V). The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronounced effects (E°' = -0.094 V), whereas in the D94V variant (E°' = -0.060 V), formation of the ferrous state is entropically disfavored. These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants.     

Myosin light chain kinase binding to plastic

Methionine-81 and/or -8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with ¹³CH₃I to produce the sulfonium ion derivatives [S-[¹³C]methylmethionine-8]glycophorin A and [S-[¹³C]methylmethionine-8 and -81]glycophorin A. ¹³C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me₄Si. A spin-lattice relaxation time of 0.4 was observed for the ¹³C-enriched methyl resonances of the sulfonium ion derivatives of Met-8 and -81, which corresponds to an effective correlation time of < 2× 10^?10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2-mercaptoethanol produces native glycophorin A which now has the ε-carbon of the methionine residue(s) 45% isotopically enriched. The ε-carbon of Met-8 was found to occur at 15.7 ppm downfield from Me₄Si whereas the ε-carbon of Met-81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me₄Si. The spin-lattice relaxation time of both resonances was found to be ～0.3 s.     

Disruption of the aspartate to heme ester linkage in human myeloperoxidase: impact on ligand binding, redox chemistry, and interconversion of redox intermediates

In human heme peroxidases the prosthetic group is covalently attached to the protein via two ester linkages between conserved glutamate and aspartate residues and modified methyl groups on pyrrole rings A and C. Here, monomeric recombinant myeloperoxidase (MPO) and the variants D94V and D94N were produced in Chinese hamster ovary cell lines. Disruption of the Asp(94) to heme ester bond decreased the one-electron reduction potential E'(0) [Fe(III)/Fe(II)] from 1 to -55 mV at pH 7.0 and 25 degrees C, whereas the kinetics of binding of low spin ligands and of compound I formation was unaffected. By contrast, in both variants rates of compound I reduction by chloride and bromide (but not iodide and thiocyanate) were substantially decreased compared with the wild-type protein. Bimolecular rates of compound II (but not compound I) reduction by ascorbate and tyrosine were slightly diminished in D94V and D94N. The presented biochemical and biophysical data suggest that the Asp(94) to heme linkage is no precondition for the autocatalytic formation of the other two covalent links found in MPO. The findings are discussed with respect to the known active site structure of MPO and its complexes with ligands.     

The intrinsic axial ligand effect on propene oxidation by horseradish peroxidase versus cytochrome P450 enzymes

The axial ligand effect on reactivity of heme enzymes is explored by means of density functional theoretical calculations of the oxidation reactions of propene by a model compound I species of horseradish peroxidase (HRP). The results are assessed vis-à-vis those of cytochrome P450 compound I. It is shown that the two enzymatic species perform C=C epoxidation and C–H hydroxylation in a multistate reactivity scenario with Fe^III and Fe^IV electromeric situations and two different spin states, doublet and quartet. However, while the HRP species preferentially keeps the iron in a low oxidation state (Fe^III), the cytochrome P450 species prefers the higher oxidation state (Fe^IV). It is found that HRP compound I has somewhat lower barriers than those obtained by the cytochrome P450 species. Furthermore, in agreement with experimental observations and studies on model systems, HRP prefers C=C epoxidation, whereas cytochrome P450 prefers C–H hydroxylation. Thus, had the compound I species of HRP been by itself, it would have been an epoxidizing agent, and at least as reactive as cytochrome P450. In the enzyme, HRP is much less reactive than cytochrome P450, presumably because HRP reactivity is limited by the access of the substrate to compound I.Electronic Supplementary Material Supplementary material is available for this article at .The paper is dedicated to D. K. Bohme on the occasion of his forthcoming 65th birthday.     

Spectroscopic description of an unusual protonated ferryl species in the catalase from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> and density functional theory calculations on related models. Consequences for the ferryl protonation state in catalase,peroxidase and chloroperoxidase

The catalase from Proteus mirabilis peroxide-resistant bacteria is one of the most efficient heme-containing catalases. It forms a relatively stable compound II. We were able to prepare samples of compound II from P. mirabilis catalase enriched in ⁵⁷Fe and to study them by spectroscopic methods. Two different forms of compound II, namely, low-pH compound II (LpH II) and high-pH compound II (HpH II), have been characterized by Mössbauer, extended X-ray absorption fine structure (EXAFS) and UV-vis absorption spectroscopies. The proportions of the two forms are pH-dependent and the pH conversion between HpH II and LpH II is irreversible. Considering (1) the Mössbauer parameters evaluated for four related models by density functional theory methods, (2) the existence of two different Fe–O_ferryl bond lengths (1.80 and 1.66 Å) compatible with our EXAFS data and (3) the pH dependence of the α band to β band intensity ratio in the absorption spectra, we attribute the LpH II compound to a protonated ferryl Fe^IV–OH complex (Fe–O approximately 1.80 Å), whereas the HpH II compound corresponds to the classic ferryl Fe^IV=O complex (Fe=O approximately 1.66 Å). The large quadrupole splitting value of LpH II (measured 2.29 mm s^?1 vs. computed 2.15 mm s^?1) compared with that of HpH II (measured 1.47 mm s^?1 vs. computed 1.46 mm s^?1) reflects the protonation of the ferryl group. The relevancy and involvement of such (Fe^IV=O/Fe^IV–OH) species in the reactivity of catalase, peroxidase and chloroperoxidase are discussed.     

The Met243 sulfonium ion linkage is responsible for the anomalous magnetic circular dichroism and optical spectral properties of myeloperoxidase

 The heme group of myeloperoxidase shows anomalous optical properties, and the enzyme possesses the unique ability to catalyze the oxidation of chloride. However, the nature of the covalently bound heme macrocycle has been difficult to identify. In this work, the electronic and magnetic properties of the heme groups in oxidized and reduced forms of wild-type and Met243Thr mutant myeloperoxidase and wild-type lactoperoxidase have been investigated using variable-temperature (1.6–273 K) magnetic circular dichroism (MCD) spectroscopy along with parallel optical absorption and electron paramagnetic resonance studies. The results provide assessment of the spin state mixtures of the oxidized and reduced samples at ambient and liquid helium temperatures and show that the anomalous MCD properties of myeloperoxidase, e.g. red-shifted and inverted signs for bands in the high-spin ferric and low-spin ferrous forms compared to other heme peroxidases and heme proteins in general, are a direct consequence of a novel electron-withdrawing sulfonium ion heme linkage involving Met243. Received: 3 May 1999 / Accepted: 9 August 1999     

Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.     

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe^V redox intermediate of myeloperoxidase, to Compound II, which has Fe^IV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.     

Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in human eosinophil peroxidase. The ester with Asp-93 is only partially formed in vivo.

The covalent heme attachment has been extensively studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage to the heavy chain is invariably present, whereas heme linkage to the light chain of EPO is present in less than one-third of EPO molecules. Mass analysis of isolated heme bispeptides supports the hypothesis of a heme b linked through two esters to the polypeptide. Mass analysis of heme monopeptides reveals that >90% have a nonderivatized methyl group at the position of the light chain linkage. Apparently, an ester had not been formed during biosynthesis. The light chain linkage could be formed by incubation with hydrogen peroxide, in accordance with a recent hypothesis of autocatalytic heme attachment based on studies with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated heme peptides after aminolysis, we unambiguously identified the acidic residues, Asp-93 of the light chain and Glu-241 of the heavy chain, that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids Permeability and 13C-nuclear magnetic resonance measurements

¹³C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of ¹³C-NMR relaxation enhancement measurements using cholesterol enriched with ¹³C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-¹³C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether-phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated ⁴⁵Ca²⁺ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether-phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for ⁴⁵Ca²⁺ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether-, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. These results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether-phosphatidylcholine bilayers, the 4-¹³C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

Localization of the disulfide bond in human antithrombin III required for heparin-accelerated thrombin inactivation

Heparin accelerates the rate of inhibition of thrombin by antithrombin III. Reduction of one of the three antithrombin disulfide bonds with dithiothreitol under mild conditions abolishes this rate-enhancing effect without affecting the rate of reaction in the absence of heparin. Alkylation of mildly reduced antithrombin III with [³H]iodacetic acid followed by digestion with cyanogen bromide yielded two major labeled peptides. The smaller peptide, containing Cys-422, was identified as extending from Gly-414 to the C-terminus, Lys-424. Our data are consistent with the larger labeled peptide being the one extending from Glu-104 to Met-243 and containing Cys-239. Cys-422 has been shown by others to be linked to Cys-239. These data indicate that the sensitive disulfide bond in antithrombin III extends between Cys-239 and Cys-422; the site at which thrombin cleaves the antithrombin III is between these two half-cystines.     

Structural similarity and functional diversity in diiron-oxo proteins

 Diiron-oxo proteins currently represent one of the most rapidly developing areas of bioinorganic chemistry. All of these proteins contain a four-helix bundle protein fold surrounding a (μ-carboxylato)diiron core, and most, if not all, of the diiron(II) sites appear to react with O₂ as part of their functional processes. Despite these common characteristics, an emerging functional diversity is one of the most striking aspects of this class of proteins. X-ray crystal structures of diiron(II) sites are now available for four of these proteins: hemerythrin (Hr), the hydroxylase protein of methane monooxygenase (MMOH), the R2 protein of Escherichia coli ribonucleotide reductase (RNR-R2), and a plant acyl-carrier protein Δ⁹-desaturase. The structure of the diiron(II) site in Hr, the sole O₂ carrier in the group, is clearly distinct from the other three, whose function is oxygen activation. The Hr diiron site is more histidine rich, and the oxygen-activating diiron sites contain a pair of (D/E)X_30–37EX₂H ligand sequence motifs, which is clearly not found in Hr. The Hr diiron site apparently permits only terminal O₂ coordination to a single iron, whereas the oxygen-activating diiron(II) centers present open or labile coordination sites on both irons of the center, and show a much greater coordinative flexibility upon oxidation to the diiron(III) state. Intermediates at the formal Fe^IIIFe^III and Fe^IVFe^IV oxidation levels for MMOH and formal Fe^IIIFe^IV oxidation level for RNR-R2 have been identified during reactions of the diiron(II) sites with O₂. An [Fe₂(μ-O)₂]^4+, 3+ "diamond core" structure has been proposed for the latter two oxidation levels. The intermediate at the Fe^IIIFe^IV oxidation level in RNR-R2 is kinetically competent to generate a stable, functionally essential tyrosyl radical. The Fe^IVFe^IV oxidation level is presumed to effect hydroxylation of hydrocarbons in MMOH, but the mechanism of this hydroxylation, particularly the involvement of discrete radicals, is currently controversial. The biological function of diiron sites in three members of this class, rubrerythrin, ferritin and bacterioferritin, remains enigmatic. Received: 31 July 1996 / Accepted: 4 October 1996     

Theoretical investigation on the oxidative chlorination performed by a biomimetic non-heme iron catalyst

The present study is a part of an effort to understand the mechanism of the oxidative chlorination, as performed by a biomimetic non-heme iron complex. This catalytically active complex is generated from a peroxide and [(TPA)Fe^IIICl₂]⁺ [TPA is tris(2-pyridylmethyl)amine]. The reaction catalyzed by [(TPA)FeCl₂]⁺/ROOH involves either [(TPA)ClFe^V=O]²⁺ or [(TPA)ClFe^IV=O]⁺ as an intermediate. On the basis of density functional theory the reaction of these two possible catalysts with cyclohexane is investigated. A question addressed is how the competing hydroxylation of the substrate is avoided. It is demonstrated that the high-valent iron complex [(TPA)Cl–Fe^V=O]²⁺ is capable of stereospecific alkane chlorination, based on an ionic rather than on a radical pathway. In contrast, the results found for [(TPA)ClFe^IV=O]⁺ cannot explain the experimental findings. In this case the transition states for chlorination and hydroxylation are energetically too close. The exclusive chlorination of the substrate by Cl–Fe^IV=O may be explained by an indirect or a direct effect, altering the position of the competing rebound barriers. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis, spectroscopy, electrochemistry, and photochemistry of cyano-bridged mixed-valence coordination compounds containing two different types of intervalent charge-transfer bands

The tetranuclear and pentanuclear mixed-valence coordination compounds Na[(NC)₅Fe^II-μ(CN)-Pt^IV(NH₃)₄-μ(NC)-Fe^II(CN)₄-μ(CN)-Ru^III(NH₃)₅], or FePtFeRu, and [Ru^III(NH₃)₅-μ(NC)-Fe^II(CN)₄-μ(CN)-Pt^IV(NH₃)₄-μ(NC)-Fe^II(CN)₄-μ(CN)-Ru^III(NH₃)₅](OSO₂CF₃)₂, or RuFePtFeRu, were synthesized and characterized by IR and UV-Vis spectroscopy, electron microprobe analysis (EPMA), inductively coupled plasma (ICP), and cyclic voltammetry (CV). Both molecules exhibit Fe^II → Pt^IV intervalent charge transfer (IVCT) absorptions in the 400-450 nm range and Fe^II → Ru^III transition(s) between 750 and 950 nm. The energies, intensities, and half-widths of these transitions correspond well with those of model compounds. The cyclic voltammogram of FePtFeRu between 0.00 and 0.90 V versus SCE exhibits two quasi-reversible Fe waves at 0.56 and 0.74 V versus SCE, while that for RuFePtFeRu has only one Fe redox event at 0.72 V versus SCE. When the potential of the working electrode is scanned negative of −0.38 V versus SCE, however, both complexes undergo an ECE (electrochemical-chemical-electrochemical) mechanism whereby the electrochemical reduction of Ru(III) is followed by a double electron transfer to reduce Pt(IV) to Pt(II). Upon reduction to Pt(II), the cyanide bridges break and the complexes dissociate into smaller fragments. Irradiation of the Fe^II → Pt^IV IVCT transition in both compounds leads to a photolysis solution that contains dissociated Fe(II)-Ru(III) as one of its products. Irradiation of the Fe^II → Ru^III IVCT transition yields a similar UV-Vis spectrum, suggesting that the same intermediate is common to both photolysis mechanisms. The implications of this research within the larger context of multiple electron transfer are also discussed.     

Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a₃/Cu_B site, and for proton pumping. When present, the side chain of Glu-242 is orientated “down” towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity “above” Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an “up” position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the “up” position is preferred energetically when the cavity contains four water molecules, but the “down” position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the “resting” state of the enzyme, and that the “up/down” equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O₂ reduction.