Coordinate High Spin Research Articles

A new inhibition mechanism in the multifunctional catalytic hemoglobin dehaloperoxidase as revealed by the DHP A(V59W) mutant: A spectroscopic and crystallographic study

As multifunctional catalytic hemoglobins, dehaloperoxidase isoenzymes A and B (DHP A and B) are among the most versatile hemoproteins in terms of activities displayed. The ability of DHP to bind over twenty different substrates in the distal pocket might appear to resemble the promiscuousness of monooxygenase enzymes, yet there are identifiable substrate-specific interactions that can steer the type of oxidation (O-atom vs. electron transfer) that occurs inside the DHP distal pocket. Here, we have investigated the DHP A(V59W) mutant in order to probe the limits of conformational flexibility in the distal pocket as it relates to the genesis of this substrate-dependent activity differentiation. The X-ray crystal structure of the metaquo DHP A(V59W) mutant (PDB 3K3U) and the V59W mutant in complex with fluoride [denoted as DHP A(V59W-F)] (PDB 7MNH) show significant mobility of the tryptophan in the distal pocket, with two parallel conformations having W59-N[Formula: see text] H-bonded to a heme-bound ligand (H2O or F[Formula: see text], and another conformation [observed only in DHP A(V59W-F)] that brings W59 sufficiently close to the heme as to preclude axial ligand binding. UV-vis and resonance Raman spectroscopic studies show that DHP A(V59W) is 5-coordinate high spin (5cHS) at pH 5 and 6-coordinate high spin (6cHS) at pH 7, whereas DHP A(V59W-F) is 6cHS from pH 5 to 7. Enzyme assays confirm robust peroxidase activity at pH 5, but complete loss of activity at pH 7. We find no evidence that tryptophan plays a role in the oxidation mechanism ([Formula: see text]. radical formation). Instead, the data reveal a new mechanism of DHP inhibition, namely a shift towards a non-reactive form by OH[Formula: see text] ligation to the heme-Fe that is strongly stabilized (presumably through H-bonding interactions) by the presence of W59 in the distal cavity.

Spectroscopic evidence for a 5-coordinate oxygenic ligated high spin ferric heme moiety in the Neisseria meningitidis hemoglobin binding receptor

BackgroundFor many pathogenic microorganisms, iron acquisition represents a significant stress during the colonization of a mammalian host. Heme is the single most abundant source of soluble iron in this environment. While the importance of iron assimilation for nearly all organisms is clear, the mechanisms by which heme is acquired and utilized by many bacterial pathogens, even those most commonly found at sites of infection, remain poorly understood. MethodsAn alternative protocol for the production and purification of the outer membrane hemoglobin receptor (HmbR) from the pathogen Neisseria meningitidis has facilitated a biophysical characterization of this outer membrane transporter by electronic absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman techniques. ResultsHmbR co-purifies with 5-coordinate high spin ferric heme bound. The heme binding site accommodates exogenous imidazole as a sixth ligand, which results in a 6-coordinate, low-spin ferric species. Both the 5- and 6-coordinate complexes are reduced by sodium hydrosulfite. Four HmbR variants with a modest decrease in binding efficiency for heme have been identified (H87C, H280A, Y282A, and Y456C). These findings are consistent with an emerging paradigm wherein the ferric iron center of bound heme is coordinated by a tyrosine ligand. ConclusionIn summary, this study provides the first spectroscopic characterization for any heme or iron transporter in Neisseria meningitidis, and suggests a coordination environment heretofore unobserved in a TonB-dependent hemin transporter. General SignificanceA detailed understanding of the nutrient acquisition pathways in common pathogens such as N. meningitidis provides a foundation for new antimicrobial strategies.

Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa

Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (K d ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.

Role of C-terminal acidic cluster in stabilization of heme spin state of ascorbate peroxidase from Leishmania major

Architecture of hemoprotein is solely responsible for different nature of heme coordination. Here we report that substitution of the acidic surface residue Glu226 to Ala in ascorbate peroxidase from Leishmania major alters the 5 coordinate high spin (5cHS) to a 6 coordinate low spin (6cLS) form at pH 7.5. Using UV-visible spectrophotometry, we show that the sixth ligand of heme in Glu226Ala at pH 7.5 is hydroxo. When the pH is decreased to 5.5, a new species of Glu226Ala appeared that had a spectrum characteristic of a 6cHS derivative. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I was not seen in the Glu226Ala in presence of H(2)O(2). Similarly guaiacol, ascorbate and ferrocytochrome c oxidation rate was 10(3) orders less for the Glu226Ala mutants compared to the wild type. These data suggested that surface acidic residue Glu226 might play role in proper maintenance of active site conformation.

Resonance Raman Probes of the Internal Binding Pocket of Dehaloperoxidase from Amphitrite ornata

Using Resonance Raman spectroscopy and activity assays we have determined that there are potent inhibitors of DHP activity and these inhibitors play a major role in the biology of DHP. Resonance Raman spectroscopy can detect substrate or inhibitor binding by monitoring shifts in the core size marker modes, ν2 and ν3, and the vinyl substituent mode, νC=C, of the heme. Para-halogenated phenols bind internally, displacing the sixth position water molecule of the iron (in agreement with X-ray crystallography) resulting in a 5-coordinate high spin heme. Tri-halogenated phenols bind externally, forcing the distal histidine into the closed conformation (also in agreement with X-ray crystallography) resulting in a 6-coordinate high spin heme. The internal and external binding sites are supported by NMR data. X-ray crystallography shows when para-halogenated phenols bind internally, the halogen plugs into a hydrophobic cavity just above the back edge of the distal pocket that is analogous to the xenon 4 binding site in Sperm whale met-myoglobin often referred to as a “packing defect.” Analysis of the X-ray crystal structures of the 4-iodo-, 4-bromo-, and 4-chlorophenol/DHP complexes show a general trend in the position of the halogenated phenol with 4-iodophenol binding farthest into the cavity followed by 4-bromophenol, and 4-chlorophenol. Using RR spectroscopy, we demonstrate that the dissociation constants of the para-halogenated phenols mimic the trend observed in X-ray crystallography with 4-iodophenol being the most tightly bound in the distal pocket. DHP clearly demonstrates the importance of the hydrophobic cavity as an inherent selector for substrates entering the distal pocket by making use of the cavity-halogen hydrophobic interaction concomitantly preventing non-halogenated substrates from binding. Using activity assays, we demonstrate that internal binding of mono-halogenated phenols inhibits the function of DHP on the externally bound tri-halogenated substrates.

Critical Role of the Heme Axial Ligand, Met95, in Locking Catalysis of the Phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O(2) or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O(2) molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O(2) complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O(2). In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O(2), CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.

Structural Diversities of Active Site in Clinical Azole-bound Forms between Sterol 14α-Demethylases (CYP51s) from Human and Mycobacterium tuberculosis

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET-bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single gx and gz values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.

Manipulating the covalent link between distal side tryptophan, tyrosine, and methionine in catalase‐peroxidases: An electronic absorption and resonance Raman study

Electronic absorption and resonance Raman spectroscopies have been applied to study the ferric and ferrous forms, and fluoride complexes of the Tyr249Phe and Met275Ile variants of the recombinant catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803. Both crystal structures and mass spectrometric analysis demonstrated that Tyr249 and Met275 are part of a novel KatG-specific covalent adduct including in addition a conserved tryptophan. Its role is not well established, but it has been shown to be essential for the catalase activity. In the present work we investigate the effect of mutation on the protein stability and ligand binding. The results clearly show that mutation weakens the heme binding to the protein, giving rise to a partial conversion from the 5-coordinate high spin of the wild-type protein to 6-coordinate low-spin heme. An internal ligand binds the heme iron on the distal side as a consequence of protein destabilization and partially prevents the binding of external ligand such as fluoride. The results are compared with those previously reported for the Trp122Ala and Trp122Phe variants.

Purification and characterization of a new cationic peroxidase from fresh flowers of Cynara scolymus L.

A basic heme peroxidase isoenzyme (AKPC) has been purified to homogeneity from artichoke flowers ( Cynara scolymus L.). The enzyme was shown to be a monomeric glycoprotein, M r=42 300±1000, (mean±S.D.) with an isoelectric point >9. The native enzyme exhibits a typical peroxidase ultraviolet–visible spectrum with a Soret peak at 404 nm ( ϵ=137 000±3000 M −1 cm −1) and a Reinheitzahl (Rz) value ( A 404nm/ A 280nm) of 3.8±0.2. The ultraviolet–visible absorption spectra of compounds I, II and III were typical of class III plant peroxidases but unlike horseradish peroxidase isoenzyme C, compound I was unstable. Resonance Raman and UV–Vis spectra of the ferric form show that between pH 5.0 and 7.0 the protein is mainly 6 coordinate high spin with a water molecule as the sixth ligand. The substrate-specificity of AKPC is characteristic of class III (guaiacol-type) peroxidases with chlorogenic and caffeic acids, that are abundant in artichoke flowers, as particularly good substrates at pH 4.5. Ferric AKPC reacts with hydrogen peroxide to yield compound I with a second-order rate constant ( k +1) of 7.4×10 5 M −1 s −1 which is significantly slower than that reported for most other class III peroxidases. The reaction of ferric and ferrous AKPC with nitric oxide showed a potential use of this enzyme for quantitative spectrophotometric determination of NO and as a component of novel NO sensitive electrodes.

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy.

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.

Spectroscopic characterization of a manganese–lignin peroxidase hybrid isozyme produced by Bjerkandera adusta in the absence of manganese: evidence of a protein centred radical by hydrogen peroxide

Electronic absorption and electron paramagnetic resonance (EPR) spectra are reported for a novel manganese–lignin peroxidase (MnLiP) hybrid isozyme produced by Bjerkandera adusta in the absence of manganese at pH 5. The room temperature absorption and the low temperature (10 K) EPR spectra indicate that the same coordination and spin states are present at both temperatures: mainly six coordinate high spin containing low percentage six coordinate low spin ferric heme, the latter probably with a bis-imidazole coordination. A protein centred radical was detected in the presence of an excess of hydrogen peroxide and assumed to be a tryptophanyl radical. The catalytic significance of this site was addressed by specific chemical modification of the tryptophan residues that revealed a marked effect on the specific activity of the enzyme. It is proposed that substrate oxidation might proceed through a long range-electron transfer process.

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase.

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.

Chlamydomonas Chloroplast Ferrous Hemoglobin: HEME POCKET STRUCTURE AND REACTIONS WITH LIGANDS

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

Endothelial nitric oxide synthase: modulations of the distal heme site produced by progressive N-terminal deletions.

cDNAs coding for bovine endothelial nitric oxide synthase (eNOS) with N-terminal deletions of 52, 91, and 105 amino acids were constructed, and the proteins were expressed in Escherichia coli and purified by affinity chromatography. All three truncated proteins bind heme and exhibit the ferrous-CO absorption maximum at 444 nm characteristic of thiolate heme ligation. Deletion of the first 52 amino acids yields a fully active dimeric protein with the same spectroscopic properties as the wild-type. The myristoylation, palmitoylation, and polyproline domains of the enzyme located in the deleted region are therefore not required for full catalytic activity. The delta91 and delta105 proteins, which exhibit altered dimerization equilibria, retain 20 and 12%, respectively, of the maximal activity. Resonance Raman and UV-vis spectroscopy indicate that, in the absence of tetrahydrobiopterin (H4B) and l-Arg, the wild-type and delta52 proteins are predominantly five coordinate high spin, whereas the delta91 and delta105 proteins are six coordinate low spin. The delta91 and delta105 mutants bind H4B, as indicated by a concomitant decrease in the low-spin component of the UV-vis spectrum, but the binding of l-Arg is extremely slow ( approximately 15 min). Dithiothreitol readily coordinates as the sixth iron ligand in the delta91 and delta105 mutants but not in the delta52 or wild-type proteins. The dithiothreitol can be completely displaced by l-Arg but not by H4B. Resonance Raman comparison of wild-type eNOS and nNOS confirms that, in the absence of H4B and l-Arg, eNOS is primarily high spin whereas nNOS is predominantly six coordinate, low spin. The results indicate that Cys-101 is not critical for the binding of H4B and imply that some of the protein residues involved in dimer formation and in preservation of active site integrity are located, probably at the monomer-monomer interface, in the N-terminal end of the protein.

Versatility of heme coordination demonstrated in a fungal peroxidase. Absorption and resonance Raman studies of Coprinus cinereus peroxidase and the Asp245-->Asn mutant at various pH values.

The pH dependence of the electronic absorption and resonance Raman (RR) spectra of FeIII and FeII forms of Coprinus cinereus peroxidase (CIP) and its Asp245-->Asn (D245N) mutant has been examined in detail. The spectral data were obtained in the pH range 3.8-12.0. These spectra were used to assess the spin and ligation states of the heme via the porphyrin marker band frequencies and the wavelengths of the absorption maxima, especially that of the band (CT1) due to the charge transfer from the porphyrin to the heme iron via the a' 2u(pi)-->eg (d pi) electronic transition. The RR spectra were obtained by using different excitation wavelengths and polarized light. The data obtained for ferric CIP show that two pH-induced structural transitions exist. At acid pH the Soret and the CT1 absorption maxima occur at 394 and 652 nm, respectively, compared with the values of 403 and 649 nm observed at neutral pH. The electronic data indicate that at acid pH the proximal Fe-Im bond might be weakened or ruptured, and the RR spectra show a new species (5-c HS) different from the normal neutral 5-coordinate high-spin (5-c HS) heme. At pH 12.0, the protein converts to a 6-coordinate low-spin (6-c LS) heme with a hydroxyl ligand coordinated in the sixth position of the heme iron and strongly hydrogen-bonded with the positively charged guanidinium group of the distal Arg51 residue. Replacement of the aspartate carboxylate group of Asp245, which acts as hydrogen-bond acceptor to the proximal His183 ligand of the heme Fe, with a carboxamide group of an asparagine residue has a profound influence on the heme coordination. The RR spectra of the Fe(II) form of this mutant at both neutral and alkaline pH values show a band at 204 cm-1 assigned to the Fe-His stretch associated with a fairly weak or non-hydrogen-bonded imidazole. The ferric form of the mutant shows a great variability in coordination and spin states upon pH titration. Between pH 8.8 and 3.8 the spectra are mainly characteristic of a 6-coordinate high-spin heme, presumably with a water molecule bound on the distal side of the Fe atom. The pKa of the alkaline transition of the mutant is much lower than that of the wild-type protein. At pH 10.0 the D245N mutant is in its final alkaline form, which markedly differs from that of the parent enzyme. The spectral data indicate that the majority of the protein has 5-coordinate high-spin heme (5-c HS), with the Fe-His 183 bond broken and the distal axial coordination site of the heme iron occupied by a hydroxyl group, which is strongly hydrogen-bonded with distal Arg51. Therefore, the Asp245-->Asn mutation on the proximal side results in the breakage of the Fe-His bond at alkaline pH.

Heme Oxygenase-2: PROPERTIES OF THE HEME COMPLEX OF THE PURIFIED TRYPTIC FRAGMENT OF RECOMBINANT HUMAN HEME OXYGENASE-2

Recombinant human microsomal heme oxygenase-2 was expressed in Escherichia coli. Tryptic digestion of the membrane fraction, in which the wild-type enzyme was localized, yielded a soluble tryptic peptide of 28 kDa, which retained the ability to accept electrons from NADPH-cytochrome P-450 reductase and the enzymatic activity for conversion of heme to biliverdin. The tryptic fragment, when purified to apparent homogeneity, bound one equivalent of heme to form a substrate-enzyme complex that had spectroscopic properties characteristic of heme proteins, such as myoglobin and hemoglobin. Optical absorption, Raman scattering, and EPR studies of the heme-tryptic fragment complex revealed that the ferric heme was six coordinate high spin at neutral pH and six coordinate low spin at alkaline pH, with a pK alpha value of 8.5. EPR and Raman scattering studies indicated that a neutral imidazole of a histidine residue served as the proximal ligand in the heme-heme oxygenase-2 fragment complex. The reaction with hydrogen peroxide converted the heme of the heme oxygenase-2 fragment complex into a verdoheme-like intermediate, while the reaction with m-chloroperbenzoic acid yielded a oxoferryl species. These spectroscopic properties are similar to those obtained for heme oxygenase-1, and thus the catalytic mechanism of heme oxygenase-2 appears to be similar to that of heme oxygenase-1.

A six-coordinate high spin protoporphyrin IX iron(III) complex as a model for ferric haemproteins: Mössbauer spectrum of bisaquo(protoporphyrinato IX)iron(III) encapsulated in aqueous detergent micelles

The Mössbauer spectrum is reported for a six- coordinate high spin iron(III) protoporphyrin complex, [(PPIX)Fe(H 2O) 2] +, which is monodispersed in an aqueous frozen solution of sodium dodecyl sulphate micelles. The isomer shift of 0.46 mm −1 and the quadrupole splitting of 1.19 mm 1̄ are similar to those of aquo met myoglobin. These results are compared with the literature results on other high spin six-coordinated models and with those of some proteins. This is the first report of a Mössbauer study on a haem encapsulated in a detergent micelle.

Synthesis and characterisation of cobalt(II), cobalt(III) and rhodium(III) complexes ofN, N?, N?, N?-tetra(2-cyanoethyl) cyclam [TCEC] andN, N?, N?, N?-tetra)3-aminopropyl) cyclam [TAPC

Complexes of cobalt(II), cobalt(III) and rhodium(III) with TCEC and TAPC have been synthesised. TCEC with cobalt(II) gave [Co(TCEC)Br]Br and [Co(TCEC)Cl]Cl, five coordinate high spin square pyramid complexes, but the corresponding cobalt(III) complex could not be characterised. Rhodium(III) gave a six coordinate [Rh(TCEC)Cl2]Cl complex, in which the two coordinated chlorides have acis-geometry and the four pendant arms lie on one side of the N4 plane with none of the —C≡N groups coordinated TAPC on the other hand gives the cobalt(III) complex, [Co(TAPC)Br]Br2, in which one of the amino groups of the four pendant arms is coordinated to cobalt. Rhodium(III) with TAPC gave [Rh(TAPC)Cl]Cl2 in which one axial site is occupied by the amino group of one of the pendant arms and the other by Cl−.

Mössbauer studies on tetra(p-sulphophenyl)porphine iron(III) solutions

The Mössbauer spectra of frozen aqueous solutions of iron(III) over the pH range 1.9 to 12 are reported. The spectra show that tetra(p-sulphophenyl)porphine iron(III) exists principally in two forms, monomeric and as μ-oxo-oligomers, depending on the pH of the solution. Above pH 4.0 only the μ-oxo-oligomer exists and at very low pHs (2.0 or less) only monomers are present. The quadrupole splitting of the monomer is the largest yet recorded for a five coordinate high spin iron(III) porphyrin.

Mössbauer studies on protoporphyrin IX iron(II) frozen solutions containing ligands that cause the iron to be in a five coordinate high spin iron(II) environment

Mössbauer parameters are presented for a number of protoporphyrin IX iron(II) complexes containing ligands that allow the iron to be in a five coordinate high spin iron(II) electronic environment. Such environments are characterised by large quadrupole splittings in the range 4.0 to 4.4 mm s −1. These compounds have characteristic electronic spectra. The implications of catechol type ligands binding protoporphyrin IX iron II/III moieties are discussed.