Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

Myeloperoxidase of the leukocyte of normal blood. Nature of the prosthetic group of myeloperoxidase

The absorption spectra of alkaline pyridine hemochrome of myeloperoxidase in its native, acid, and modified forms were similar to those of heme a, and the molar extinction coefficient of myeloperoxidase heme was very similar to that of heme a, assuming that myeloperoxidase contains only one heme. The anaerobic titration of myeloperoxidase with dithionite showed that one electron was consumed per molecule of the enzyme for its conversion to its reduced form. The EPR spectrum of myeloperoxidase indicated that the enzyme contains both high-spin heme and non-heme iron. Carbonyl reagents, such as borohydride, hydrazine, and benzhydrazide, reacted with myeloperoxidase, causing blue shifts in its absorption spectrum. The heme was labeled with a tritium of boro[3H]hydride, suggesting that the reagents reacted with a formyl group on the porphyrin ring of the myeloperoxidase heme. When hydrazine was added to cyanide complex I of myeloperoxidase the complex was converted to the hydrazine-enzyme compound. Myeloperoxidase reacted with bisulfite to form a compound with an absorption spectrum similar to that of cyanide complex I. Borohydride-treated myeloperoxidase formed only one cyanide complex, while the native enzyme formed two different cyanide complexes, I (Kd = 0.3 muM) and II (approximate Kd = 0.1 mM). The EPR spectrum indicated that cyanide complex I of myeloperoxidase still contained high-spin heme. The results suggested that cyanide complex I and the bisulfite compound of myeloperoxidase were adducts between the nucleophilic reagents and the formyl group of myeloperoxidase heme. Based on these results, we concluded that one of the two iron atoms in a myeloperoxidase molecule exists in a formyl-heme moiety similar to heme a and the other exists as a non-heme iron.     

Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent

Soret excitation resonance Raman spectroscopy has been used to characterize dimeric human leukocyte myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) and monomeric bovine spleen green haemoprotein. The spectra of the two proteins, under the same conditions of iron valence and ligation, are essentially identical. Owing to strong symmetry reduction effects, the spectra are more complex than usually observed for haemoproteins. It is possible, however, to assign the high-frequency vibrations and, from these assignments, to determine structural features of the iron chromophores. In the resting protein, the iron adopts a six-coordinate high-spin configuration in both proteins; cyanide addition produces six-coordinate low-spin species, and in the ferrous enzymes the iron appears to be five-coordinate and high-spin. The proteins are stable to laser excitation and do not photoreduce under illumination. No evidence is found for unusual peripheral substituents, such as formyl or protonated Schiff's base group, in conjugation with the main chromophore in the native protein. The vibrational data are consistent with an iron chlorin chromophore, although other electronic effects, in addition to those produced by porphyrin ring reduction, are necessary to account for the optical properties of the proteins. The similarity in Raman spectra for myeloperoxidase and green haemoprotein indicates that the two iron sites in myeloperoxidase are equivalent.     

Resonance Raman studies of CuA-modified cytochrome c oxidase

Modification of the CuA site in mammalian cytochrome c oxidase has been used to elucidate the functional role of this center in the catalytic cycle of the enzyme. Both heat treatment in detergents and chemical modification by p-(hydroxymercuri)benzoate (pHMB) convert CuA to a lower potential type II center and effectively remove the site from the electron-transfer pathway during turnover. In this study, resonance Raman spectroscopy has been employed to investigate the effects of these CuA modifications on the heme active sites. The Raman data indicate some environmental perturbation of the heme a3 chromophore in the modified derivatives. Only pHMB modification and SB-12 heat treatment produced significant effects in the Raman spectra of the fully reduced enzyme. These perturbations are much less evident in the spectra obtained within 10 ns of CO photolysis from the fully reduced species of the modified enzymes. Transient Raman studies further indicate that the half-time for CO religation in the modified enzymes is quite similar to that of the native protein.     

Human myeloperoxidase: structure of a cyanide complex and its interaction with bromide and thiocyanate substrates at 1.9 A resolution

The 1.9 A X-ray crystal structure of human myeloperoxidase complexed with cyanide (R = 0.175, R(free) = 0.215) indicates that cyanide binds to the heme iron with a bent Fe-C-N angle of approximately 157 degrees, and binding is accompanied by movement of the iron atom by 0.2 A into the porphyrin plane. The bent orientation of the cyanide allows the formation of three hydrogen bonds between its nitrogen atom and the distal histidine as well as two water molecules in the distal cavity. The 1.85 A X-ray crystal structure of an inhibitory complex with thiocyanate (R = 0.178, R(free) = 0.210) indicates replacement of chloride at a proximal helix halide binding site in addition to binding in the distal cavity in an orientation parallel with the heme. The thiocyanate replaces two water molecules in the distal cavity and is hydrogen bonded to Gln 91. The 1.9 A structures of the complexes formed by bromide (R = 0.215, R(free) = 0.270) and thiocyanate (R = 0.198, R(free) = 0.224) with the cyanide complex of myeloperoxidase show how the presence of bound cyanide alters the binding site for bromide in the distal heme cavity, while having little effect on thiocyanate binding. These results support a model for a single common binding site for halides and thiocyanate as substrates or as inhibitors near the delta-meso carbon of the porphyrin ring in myeloperoxidase.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Structure of Helicobacter pylori catalase, with and without formic acid bound, at 1.6 A resolution

Helicobacter pylori produces one monofunctional catalase, encoded by katA (hp0875). The crystal structure of H. pylori catalase (HPC) has been determined and refined at 1.6 A with crystallographic agreement factors R and R(free) of 17.4 and 21.9%, respectively. The crystal exhibits P2(1)2(1)2 space group symmetry and contains two protein subunits in the asymmetric unit. The core structure of the HPC subunit, including the disposition of a heme b prosthetic group, is closely related to those of other catalases, although it appears to be the only clade III catalase that has been characterized that does not bind NADPH. The heme iron in one subunit of the native enzyme appears to be covalently modified, possibly with a perhydroxy or dioxygen group in a compound III-like structure. Formic acid is known to bind in the active site of catalases, promoting the breakdown of reaction intermediates compound I and compound II. The structure of an HPC crystal soaked with sodium formate at pH 5.6 has also been determined to 1.6 A (with R and R(free) values of 18.1 and 20.7%, respectively), revealing at least 36 separate formate or formic acid residues in the HPC dimer. In turn, the number of water molecules refined into the models decreased from 1016 in the native enzyme to 938 in the formate-treated enzyme. Extra density, interpreted as azide, is found in a location of both structures that involves interaction with all four subunits in the tetramer. Electron paramagnetic resonance spectra confirm that azide does not bind as a ligand of the iron and that formate does bind in the heme pocket. The stability of the formate or formic acid molecule found inside the heme distal pocket has been investigated by calculations based on density functional theory.     

The interaction of myeloperoxidase with ligands as studied by EPR

1. The reaction of myeloperoxidase with fluoride, chloride and azide has been studied by EPR. 2. Fluoride decreases the rhombicity of the high-spin heme signal of myeloperoxidase and the nuclear spin of the fluoride atom induces a splitting in g parallel of 35 G. This observation demonstrates that fluoride binds as an axial ligand to the heme iron of the enzyme. 3. Addition of chloride to the fluoride-treated enzyme increases the rhombicity of the high-spin heme signal and brings about a disappearance of the splitting at g parallel. The addition of azide to the fluoride-treated enzyme changes the spin state of the heme iron from a high-to a low-spin state (gx = 2.68, gy = 2.22 and gz = 1.80). 4. Upon addition of chloride or fluoride to low-spin azido-myeloperoxidase this compound is converted into the high-spin chlorido- or fluorido-myeloperoxidase. These observations demonstrate that these ligands compete for a binding site at or close to the heme iron of myeloperoxidase.     

Effect of sulfhydryl group modification on the activity of bovine ferrochelatase

The role of sulfhydryl groups in the activity of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), has been examined by using a variety of sulfhydryl group-specific reagents. The enzyme is rapidly inactivated in a pseudo-first order reaction by N-ethylmaleimide and monobromobimane and more slowly by iodoacetamide and bromotrimethylammoniobimane. Reaction with [3H]N-ethylmaleimide indicates that modification of a single sulfhydryl group is sufficient to inactivate bovine ferrochelatase. The enzyme is protected from inactivation by one substrate, ferrous iron, but not by the porphyrin substrate. Mercury and arsenite are reversible inhibitors. The fluorescence of the bound bimane is blue shifted 8 nm from that obtained in aqueous solutions and is sensitive to quenching by iodide.     

An EPR study of myeloperoxidase in human granulocytes.

1. EPR spectra of human granulocytes (4 - 10(8) cells per ml) show an intense high-spin ferric heme signal with rhombic symmetry (gx = 6.90 and gy = 5.07) for the heme group. These g-values are identical to those of partially purified myeloperoxidase and thus the signal is derived from ferric myeloperoxidase. In chicken granulocytes, which contain little or no myeloperoxidase, only an axial type of heme iron signal, weak in intensity, can be detected at g = 6.0. 2. Upon phagocytosis of latex particles by human granulocytes the high-spin heme signal with rhombic symmetry is slowly converted into a signal with axial symmetry (gx = gy = 6.0), showing that the EPR signals of myeloperoxidase in the intact cell can be used to study the involvement of the enzyme in metabolic changes during phagocytosis.     

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.     

Studies on the magneto-optical rotation of porphyrins, hemins and methemoglobin compounds]

Using the method of magneto-optical rotation (MOR) various porphyrin derivatives, hemin and heme compounds, and a number of methemoglobin complexes were investigated. The spectra were recorded from 450-600 nm; with methemoglobin also in the Soret region. 1. The metalfree porphyrin derivatives (deutero-, meso-, hemato- and protoporphyrins) were measured in strongly acidic aqueous solution. The derivatives thus present as di-cations yield highly resolved MORspectra, where the Q-bands (Oo leads to; Oo leads to 1) originated from the pi-pi transitions of the porphyrin display the curve shape characteristic of an A-term, this proving the presence of the D4h symmetry. An exception is the protoporphyrin, in which the pi-electron system of the porphyrin is perturbed by the influence of pi-electrons of the vinyl group, causing poor resolution, line broadening, and shift of the Q-bands into the lower-energy spectral region. 2. With iron porphyrins (hemin, heme and their complexes) the charge of the iron and the nature of axial ligands determine the position and intensity of the O-bands in the MOR spectrum. Low-spin complexes have a higher symmetry than the high-spin complexes. Whereas with hemin (S = 5/2), the iron located outside the heme plane strongly disturbs the porphyrin pi-system, the high symmetry of porphyrin is greatly retained in the case of heme. This can be explained by the enhanced binding distance between the bivalent iron and the porphyrin to great for a strong coupling between the microsymmetry of the iron and the macrosymmetry of the porphyrin pi-system.     

Bovine ferrochelatase. Kinetic analysis of inhibition by N-methylprotoporphyrin, manganese, and heme

The terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase EC 4.99.1.1), has been purified to apparent homogeneity from bovine liver mitochondria using a scheme similar to that reported by Taketani and Tokunaga (Taketani, S. and Tokunaga, R. (1981) J. Biol. Chem. 256, 12748-12753) for purification of the enzyme from rat liver. The final yield was 49% with a 2000-fold purification. Ferrochelatase has an apparent molecular weight of approximately 40,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and column chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. The purified enzyme was only slightly stimulated by added lipid and was inhibited by Mn2+, Pb2+, and Hg2+. Bovine ferrochelatase utilized proto-, meso-, and deuteroporphyrin, but not disubstituted porphyrins (2,4-disulfonic and 2,4-bisglycol deuteroporphyrin). N-Methylprotoporphyrin, a toxic by-product of the metabolism of some drugs, was found to inhibit ferrochelatase in a competitive fashion with respect to porphyrin with a Ki of 7 nM and uncompetitive with respect to iron. Manganese inhibits ferrochelatase competitively with respect to iron (Ki = 15 microM) and noncompetitively with respect to the porphyrin substrate. Heme, one of the products, is a noncompetitive inhibitor with respect to iron. These findings lead to a sequential Bi Bi kinetic model for ferrochelatase with iron binding occurring prior to porphyrin binding and heme being released prior to the release of two protons.     

Porphyrin-mediated cell surface heme capture from hemoglobin by Porphyromonas gingivalis

The porphyrin requirements for growth recovery of Porphyromonas gingivalis in heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as for Haemophilus influenzae, reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain of P. gingivalis recognizes heme by a mechanism that is solely porphyrin mediated.     

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     

Iron normal mode dynamics in (nitrosyl)iron(II)tetraphenylporphyrin from X-ray nuclear resonance data

The complete iron atom vibrational spectrum has been obtained by refinement of normal mode calculations to nuclear inelastic x-ray absorption data from (nitrosyl)iron(II)tetraphenylporphyrin, FeTPP(NO), a useful model for heme dynamics in myoglobin and other heme proteins. Nuclear resonance vibrational spectroscopy (NRVS) provides a direct measurement of the frequency and iron amplitude for all normal modes involving significant displacement of (57)Fe. The NRVS measurements on isotopically enriched single crystals permit determination of heme in-plane and out-of-plane modes. Excellent agreement between the calculated and experimental values of frequency and iron amplitude for each mode is achieved by a force-field refinement. Significantly, we find that the presence of the phenyl groups and the NO ligand leads to substantial mixing of the porphyrin core modes. This first picture of the entire iron vibrational density of states for a porphyrin compound provides an improved model for the role of iron atom dynamics in the biological functioning of heme proteins.     

High resolution proton nuclear magnetic resonance spectroscopy of lactoperoxidase

The first high resolution proton nuclear magnetic resonance spectra are reported for the native ferric and ferric cyano complexes of bovine lactoperoxidase. The spectrum of the native species exhibits broad heme signals in a far downfield region characteristic of the high-spin ferric state. The low-spin cyano complex yields a proton nuclear magnetic resonance spectrum with signals as far as 68.5 ppm downfield and as far as -28 ppm upfield of the tetramethylsilane reference. These peak positions are anomalous with respect to those seen only as far as 35 ppm downfield in other cyano hemoprotein complexes. An extreme asymmetry in the unpaired spin delocalization pattern of the iron porphyrin is suggested. The unusual proton nuclear magnetic resonance properties parallel distinctive optical spectral properties and the exceptional resistance to heme displacement from the enzyme. Lactoperoxidase utilized in these studies was isolated from raw milk and purified by an improved, rapid chromatographic procedure.     

Structure of a stable form of sulfheme

A stable green heme was extracted from ferric cyanosulfmyoglobin after it had undergone an internal conversion reaction. After iron removal and conversion to the methyl ester, the resulting green porphyrin was purified by high-pressure liquid chromatography. Visible, 1H NMR, and mass spectrometric studies provided evidence to identify the substituents of the porphyrin. Nuclear Overhauser enhancements enabled an assignment of the single modified pyrrole. Substituent positions 1, 2, 5, 6, 7, and 8 have the original protoporphyrin IX substituents. At ring B, the 4-vinyl group has cyclized with a single sulfur atom to form a fifth ring with a 2,5-dihydrothiophene type of structure.     

A new thioether-ligated iron porphyrin as a model of a protonated form of P450 active site

Thioether-ligated iron porphyrin (complex 1) was synthesized as a model of the protonated form of P450 to explore the possible involvement of the protonated form in the catalytic cycle, and ether-ligated iron porphyrin (complex 2) was also synthesized for comparison. The thioether and ether ligands enhanced heterolytic O-O bond cleavage of peroxy acid-iron porphyrin complex even in highly hydrophobic media without the assistance of acid or base, using mCPPAA as an oxidant. Competitive oxidation of cyclooctane/cyclooctene catalyzed by iron porphyrins showed that complexes 1 and 2 are less effective than heme thiolate (P450 and a synthetic heme thiolate (SR complex)) in oxidizing alkane. The possibility that thiol-ligated heme, which is a protonated form of heme thiolate, is not involved in the active intermediate structure of P450 is indicated by this result. This is the first report concerning the oxidizing ability of a thioether-ligated iron porphyrin.     

The role of arginyl residues in porphyrin binding to ferrochelatase

The role of cationic amino acid residues in the binding of porphyrin substrates by purified bovine ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) have been examined via chemical modification with camphorquinone-10-sulfonic acid, phenylglyoxal, butanedione, and trinitrobenzene sulfonate. The data obtained show that modification of arginyl, but not lysyl, residues results in the rapid inactivation of ferrochelatase. The 2,4-disulfonate deuteroporphyrin, which is a competitive inhibitor of mammalian ferrochelatase, protects the enzyme against inactivation. Ferrous iron has no protective effect. Reaction with radiolabeled phenylglyoxal shows that modification of 1 arginyl residue causes maximum inhibition of enzyme activity. The inactivation does not follow simple pseudo-first order reaction kinetics, but is distinctly biphasic in nature. Comparison of the enzyme kinetics for modified versus unmodified enzyme show that modification with camphorquinone-10-sulfonic acid has no effect on the Km for iron but does alter the Km for porphyrin.