Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

Effects of cyanogen bromide modification of the distal histidine on the spectroscopic and ligand binding properties of myoglobin: magnetic circular dichroism spectroscopy as a probe of distal water ligation in ferric high-spin histidine-bound heme proteins

Magnetic circular dichroism (MCD) spectroscopy has been utilized to characterize the change in coordination structure in native ferric sperm whale myoglobin upon cyanogen bromide-modification. Comparison of the MCD properties of the ferric high-spin state of cyanogen bromide-modified myoglobin (BrCN-Mb) with those of native ferric horseradish peroxidase and Aplysia myoglobin suggests that ferric BrCN-Mb is a potential MCD model for the pentacoordinate state of ferric high-spin histidine-ligated heme proteins. These five-coordinate heme proteins afford a relatively weak and unsymmetric signal in the Soret region of the MCD spectrum. In contrast, native ferric myoglobin and the benzohydroxamic acid adduct of ferric horseradish peroxidase show a strong and symmetric derivative-shaped Soret MCD signal which is indicative of hexacoordination with water and histidine axial ligands. Therefore it seems that MCD spectroscopy could be used to probe the presence of water ligated to the distal side of ferric high-spin heme proteins. The MCD spectra of the ferric-azide, ferrous-deoxy and ferrous-CO forms of BrCN-Mb have also been measured and compared to those of analogous native myoglobin complexes. The present MCD study has been extended to include new ligands, NO, thiocyanate and cyanate, which bind to ferric BrCN-Mb. With exogenous ligands such as CO, NO and thiocyanate, the coordination structures of the BrCN-Mb complexes are similar to those of the respective native myoglobin adducts. In the case of ferrous-deoxy and ferric-cyanate BrCN-Mb, however, the altered MCD spectra (and EPR for the latter) reveal changes in electronic structure which likely correlate with alterations of the coordination environment of these BrCN-Mb derivatives. Data are also presented which support the proposed tetrazole-bound structure for azide-treated BrCN-Mb (Hori, H., Fujii, M., Shiro, Y., Iizuka, T., Adachi, S. and Morishima, I. (1989) J. Biol. Chem. 264, 5715-5719) and the inability of the distal histidine of BrCN-Mb to stabilize the ferric ligand-bound state.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Assignment of the heme axial ligand(s) for the ferric myoglobin (H93G) and heme oxygenase (H25A) cavity mutants as oxygen donors using magnetic circular dichroism.

UV-visible absorption and magnetic circular dichroism (MCD) data are reported for the cavity mutants of sperm whale H93G myoglobin and human H25A heme oxygenase in their ferric states at 4 degreesC. Detailed spectral analyses of H93G myoglobin reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0, and a mixture of species at pH 7.0 including five-coordinate hydroxide-bound, and six-coordinate structures. The five-coordinate aquo structure at pH 5 is supported by spectral similarity to acidic horseradish peroxidase (pH 3.1), whose MCD data are reported herein for the first time, and acidic myoglobin (pH 3.4), whose structures have been previously assigned by resonance Raman spectroscopy. The five-coordinate hydroxide structure at pH 10.0 is supported by MCD and resonance Raman data obtained here and by comparison with those of other known five-coordinate oxygen donor complexes. In particular, the MCD spectrum of alkaline ferric H93G myoglobin is strikingly similar to that of ferric tyrosinate-ligated human H93Y myoglobin, whose MCD data are reported herein for the first time, and that of the methoxide adduct of ferric protoporphyrin IX dimethyl ester (FeIIIPPIXDME). Analysis of the spectral data for ferric H25A heme oxygenase at neutral pH in the context of the spectra of other five-coordinate ferric heme complexes with proximal oxygen donor ligands, in particular the p-nitrophenolate and acetate adducts of FeIIIPPIXDME, is most consistent with ligation by a carboxylate group of a nearby glutamyl (or aspartic) acid residue.     

The porphyrin-sensitized photooxidation of horseradish apoperoxidase.

Horseradish apoperoxidase (apoHRP) was reconstituted with various porphyrin derivatives, e.g., ferric, cupric, manganese, and zinc protoporphyrin IX, metal-free protoporphyrin IX, hematoporphyrin IX and deuteroporphyrin IX. The visible absorption spectra of these porphyrin-apoHRP complexes were examined. The time required for maximum development of the new Soret peak after reconstitution was used to measure the rate of porphyrin-apoHRP reconstitution. All of the four metal-protoporphyrins reconstituted with apoHRP at the same rate as metal-free protoporphyrin IX, whereas, for the metal-free porphyrins, the rates of reconstitution were in the order of deuteroporphyrin IX > hematoporphyrin IX > protoporphyrin IX. The porphyrins on the reconstituted porphyrin-apoHRP complexes were used as localized photosensitizers for photodynamic studies. No amino acid residues were oxidized on illumination of the ferric, cupric and manganese protoporphyrin IX-apoHRP complexes due to the paramagnetic properties of these metal ions. With diamagnetic zinc ion, two histidine and one methionine residues were oxidized which was the same as in the protoporphyrin IX- and hematoporphyrin IX-apoHRP complexes. However, only one histidine was destroyed on illumination of the deuteroporphyrin IX-apoHRP complex. The results confirmed the resistance of horseradish peroxidase to photodynamic action and suggested the involvement of at least one histidine residue in the heme environment of horseradish peroxidase.     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protoporphyrin IX-induced structural and functional changes in human red blood cells,haemoglobin and myoglobin

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin: protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

Influence of heme-surrounding amino acid residues on the manganese (V)-nitrido bond in manganese-substituted hemoproteins: resonance Raman evidence for porphyrin core expansion and reduction of the manganese(V)-nitrido stretching force constant

Nitridomanganese(V) protoporphyrin IX was prepared by hypochlorite oxidation of the corresponding manganese(III) protoporphyrin IX derivative in the presence of ammonium ion and by photolysis of the corresponding azidomanganese(III) complex. Myoglobin and horseradish peroxidase containing this novel protoporphyrin derivative were prepared for the first time. These remarkably stable species were examined by electronic absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. The MnV-N stretching modes of the nitridomanganese(V)-substituted myoglobin and horseradish peroxidase were observed at 1010 and 1003 cm-1, respectively, by resonance Raman spectroscopy, while the MnV-N stretching frequency for nitridomanganese(V) protoporphyrin IX in 0.1 N aqueous NaOH was found at 1046 cm-1. The equilibrium dissociation energies of MnV-N bonds in these complexes were estimated from vibrational overtone spacings by introducing the Morse potential energy function, were found to be around 4.5 eV, and seemed independent of the surroundings of the manganese porphyrin, although its force constant decreased from 7.3 to 6.7 mdyn/A upon incorporation into apoprotein. The porphyrin ring modes of these nitridomanganese(V) derivatives were influenced greatly upon incorporation into apoproteins, suggestive of the occurrence of porphyrin core expansion. Upon this core expansion the MnV center moves into the mean plane of porphyrin plane, but the access of nitrido (N) toward MnV is restricted due to a steric hindrance from porphyrin pyrrole nitrogens. The resulting stretched MnV-N bond might cause lowering of the MnV-N stretching frequency upon incorporation into apoprotein.     

Fluorescence study of the conformational properties of myoglobin structure. 3. pH-dependent changes in porphyrin and tryptophan fluorescence of the complex of sperm whale apomyoglobin with protoporphyrin IX; the role of the porphyrin macrocycle and iron in formation of native myoglobin structure

The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5-10.8 but change significantly at pH less than 5.5 and pH greater than 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH less than 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio I325/I350 which diminishes at pH greater than 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintenance of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.     

Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4 degrees C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP (MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met-Lys-Thr (MKT) at the N-terminus, CCP (MKT) in the presence of 60% glycerol, lyophilized YCCP, and alkaline CCP (MKT). The present study demonstrates that, while having similar electronic absorption spectra, the MCD spectra of ligand-free ferric YCCP and CCP (MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/water hexa-coordinate). The electronic absorption spectrum of ferric CCP (MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (< or = 5%) in the spectrum of CCP (MKT) from a hexa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (lambda CT = 637 nm) contains considerably increased amounts of hexa-coordinate low-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP (MKT) (lambda CT = 637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

The active site structure of E. coli HPII catalase. Evidence favoring coordination of a tyrosinate proximal ligand to the chlorin iron.

E. coli produces 2 catalases known as HPI and HPII. While the heme prosthetic group of the HPII catalase has been established to be a dihydroporphyrin or chlorin, the identity of the proximal ligand to the iron has not been addressed. The magnetic circular dichroism (MCD) spectrum of native ferric HPII catalase is very similar to those of a 5-coordinate phenolate-ligated ferric chlorin complex, a model for tyrosinate proximal ligation, as well as of chlorin-reconstituted ferric horseradish peroxidase, a model for 5-coordinate histidine ligation. However, further MCD comparisons of chlorin-reconstituted myoglobin with parallel ligand-bound adducts of the catalase clearly rule out histidine ligation in the latter, leaving tyrosinate as the best candidate for the proximal ligand.     

Chemical nature of the porphyrin pi cation radical in horseradish peroxidase compound I

The electron paramagnetic resonance (EPR) and M?ssbauer properties of native horseradish peroxidase have been compared with those of a synthetic derivative of the enzyme in which a mesohemin residue replaces the natural iron protoporphyrin IX heme prosthetic group. The oxyferryl pi cation radical intermediate, compound I, has been formed from both the native and synthetic enzyme, and the magnetic properties of both intermediates have been examined. The optical absorption characteristics of compound I prepared from mesoheme-substituted horseradish peroxidase are different from those of the compound I prepared from native enzyme [DiNello, R. K., & Dolphin, D. (1981) J. Biol. Chem. 256, 6903-6912]. By analogy to model-compound studies, it has been suggested that these optical absorption differences are due to the formation of an A2u and an A1u pi cation radical species, respectively. However, the EPR and M?ssbauer properties of the native and synthetic enzyme and of their oxidized intermediates are quite similar, if not identical, and the data favor an A2u radical for both compounds I.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.