Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine

The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.     

The heme complex of Hmu O, a bacterial heme degradation enzyme from Corynebacterium diphtheriae. Structure of the catalytic site.

Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart.     

Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O, a heme degradation enzyme in the pathogen Corynebacterium diphtheriae, catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. A bacterial expression system using a synthetic gene coding for the 215-amino acid, full-length Hmu O has been constructed. Expressed at very high levels in Escherichia coli BL21, the enzyme binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converts hemin to biliverdin with alpha-hydroxyhemin and verdoheme as intermediates. The overall conversion rate to biliverdin is approximately 4-fold slower than that by rat heme oxygenase (HO) isoform 1. Reaction of the hemin-Hmu O complex with hydrogen peroxide yields a verdoheme species, the recovery of which is much less compared with rat HO-1. Reaction of the hemin complex with meta-chloroperbenzoic acid generates a ferryl oxo species. Thus, the catalytic intermediate species and the nature of the active form in the first oxygenation step of Hmu O appear to be similar to those of the mammalian HO. However, the considerably slow catalytic rate and low level of verdoheme recovery in the hydrogen peroxide reaction suggest that the active-site structure of Hmu O is different from that of its mammalian counterpart.     

Resonance Raman studies of iron spin and axial coordination in distal pocket mutants of ferric myoglobin

We have used resonance Raman spectroscopy to study 11 distal pocket mutants and the "wild type" and native ferric sperm whale myoglobin. The characteristic Raman core-size markers v4, v3, v2, and v10 are utilized to assign the spin and coordination state of each sample. It is demonstrated that replacements of the distal and proximal histidines can discriminate against H2O as a sixth ligand and favor a pentacoordinate Fe3+ atom. Soret absorption band blueshifts are correlated with the pentacoordinate heme environment. One E7 replacement (Arg) leads to an iron spin state change and produces a low spin species. The Glu and Ala mutations at position E11 leave the protein's spin and coordination unaltered. A laser-induced photoreduction effect is observed in all pentacoordinate mutants and seems to be correlated with the loss of the heme-bound water molecule.     

Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase

Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.     

Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O is a 24-kDa soluble bacterial heme degradation enzyme found in the pathogen Corynebacterium diphtheriae, the causative agent of diphtheria. Similar to the mammalian heme oxygenase, it binds hemin stoichiometrically and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Iron is an essential nutrient for bacteria and especially important for pathogenesis. Here we report the first crystallization and preliminary crystallographic study of the heme-Hmu O complex formed from hemin and a recombinant Hmu O, which was expressed in Escherichia coli from a synthetic gene based on the putative hmu O gene sequence. Crystals of the heme-Hmu O complex were obtained by the sitting drop vapor diffusion method using a precipitant solution containing 18% (w/v) PEG 8000 and 0.2 M calcium acetate in 0.1 M sodium cacodylate (pH 6.5). Using synchrotron radiation, the heme-Hmu O crystal diffracted to 2.8 A resolution. It belongs to the monoclinic space group C2, with unit cell parameters a = 123.18 A, b = 44.51 A, c = 92.10 A, and beta = 123.3 degrees. Assuming one molecule of the heme-Hmu O complex per asymmetric unit, the calculated value of Vm is 2.89 A3/Da.     

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.     

Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the nu(Fe-His) stretching frequency appears at 221 cm(-1), which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the nu(Fe-CO) stretching frequency was detected at 521 and 494 cm(-1) in WT, M69L, and K67T, while only the 494 cm(-1) component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm(-1) component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin.     

Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release

HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae

Crystal structures of the ferric and ferrous heme complexes of HmuO, a 24-kDa heme oxygenase of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 A resolution, respectively. The HmuO structures show that the heme group is closely sandwiched between the proximal and distal helices. The imidazole group of His-20 is the proximal heme ligand, which closely eclipses the beta- and delta-meso axis of the porphyrin ring. A long range hydrogen bonding network is present, connecting the iron-bound water ligand to the solvent water molecule. This enables proton transfer from the solvent to the catalytic site, where the oxygen activation occurs. In comparison to the ferric complex, the proximal and distal helices move closer to the heme plane in the ferrous complex. Together with the kinked distal helix, this movement leaves only the alpha-meso carbon atom accessible to the iron-bound dioxygen. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediate by preventing premature heterolytic O-O bond cleavage. This allows the enzyme to oxygenate selectively at the alpha-meso carbon in HmuO catalysis.     

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Magnetic resonance spectral characterization of the heme active site of Coprinus cinereus peroxidase

Examination of the peroxidase isolated from the inkcap Basidiomycete Coprinus cinereus shows that the 42,000-dalton enzyme contains a protoheme IX prosthetic group. Reactivity assays and the electronic absorption spectra of native Coprinus peroxidase and several of its ligand complexes indicate that this enzyme has characteristics similar to those reported for horseradish peroxidase. In this paper, we characterize the H2O2-oxidized forms of Coprinus peroxidase compounds I, II, and III by electronic absorption and magnetic resonance spectroscopies. Electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) studies of this Coprinus peroxidase indicate the presence of high-spin Fe(III) in the native protein and a number of differences between the heme site of Coprinus peroxidase and horseradish peroxidase. Carbon-13 (of the ferrous CO adduct) and nitrogen-15 (of the cyanide complex) NMR studies together with proton NMR studies of the native and cyanide-complexed Coprinus peroxidase are consistent with coordination of a proximal histidine ligand. The EPR spectrum of the ferrous NO complex is also reported. Protein reconstitution with deuterated hemin has facilitated the assignment of the heme methyl resonances in the proton NMR spectrum.     

Characterization of the heme environmental structure of cytoglobin,a fourth globin in humans

Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E' of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the nu(Fe-CO) and nu(C-O) values in the resonance Raman and infrared spectra of the ferrous-CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The nu(Fe-O2) in the resonance Raman spectra of oxy Cgb can be observed at 572 cm(-1), suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.     

Investigations of photolysis and rebinding kinetics in myoglobin using proximal ligand replacements

We use laser flash photolysis and time-resolved Raman spectroscopy of CO-bound H93G myoglobin (Mb) mutants to study the influence of the proximal ligand on the CO rebinding kinetics. In H93G mutants, where the proximal linkage with the protein is eliminated and the heme can bind exogenous ligands (e.g., imidazole, 4-bromoimidazole, pyridine, or dibromopyridine), we observe significant effects on the CO rebinding kinetics in the 10 ns to 10 ms time window. Resonance Raman spectra of the various H93G Mb complexes are also presented to aid in the interpretation of the kinetic results. For CO-bound H93G(dibromopyridine), we observe a rapid large-amplitude geminate phase with a fundamental CO rebinding rate that is approximately 45 times faster than for wild-type MbCO at 293 K. The absence of an iron proximal ligand vibrational mode in the 10 ns photoproduct Raman spectrum of CO-bound H93G(dibromopyridine) supports the hypothesis that proximal ligation has a significant influence on the kinetics of diatomic ligand binding to the heme.