Conformational differences in Mycobacterium tuberculosis catalase-peroxidase KatG and its S315T mutant revealed by resonance Raman spectroscopy

KatG from Mycobacterium tuberculosis is a heme-containing catalase-peroxidase, which belongs to the class I peroxidases and is important for activation of the prodrug isoniazid (INH), a front-line antituberculosis drug. In many clinical isolates, resistance to INH has been linked to mutations on the katG gene, and the most prevalent mutation, S315T, suggests that modification of the heme pocket has occurred. Electronic absorption and resonance Raman spectra of ferric wild-type (WT) KatG and its INH-resistant mutant KatG(S315T) at different pH values and their complexes with INH and benzohydroxamic acid (BHA) are reported. At neutral pH, a quantum mechanically mixed spin state (QS) is revealed, which coexists with five-coordinate and six-coordinate high-spin hemes in WT KatG. The QS heme is the major species in KatG(S315T). Addition of either INH or BHA to KatG induces only minor changes in the resonance Raman spectra, indicating that both compounds do not directly interact with the heme iron. New vibrational modes are observed at 430, 473, and 521 cm(-1), and these modes are indicative of a change in conformation in the KatG heme pocket. The intensity of these modes and the relative population of the QS heme are stable in KatG(S315T) but not in the WT enzyme. This indicates that there are differences in heme pocket stability between WT KatG and KatG(S315T). We will discuss the stabilization of the QS heme and propose a model for the inhibition of INH oxidation by KatG(S315T).     

Mercuric chloride-induced spin or ligation state changes in ferric or ferrous human cystathionine beta-synthase inhibit enzyme activity.

Cystathionine beta-synthase is a key heme and pyridoxal phosphate-dependent enzyme involved in homocysteine metabolism in humans. The role of the recently discovered heme in this protein remains an important open question. The axial ligands to the heme in both the ferrous and ferric states have been assigned as cysteine and histidine residues, respectively. In this study, we have examined the effect of ligation and spin state changes in the heme on the activity of the enzyme. Treatment of the ferric enzyme with HgCl2 results in the conversion of six-coordinate low-spin heme to five-coordinate high-spin heme and is paralleled by a loss of activity. In contrast, treatment of the ferrous enzyme with HgCl2 results in replacement of the cysteine ligand by an unidentified sixth ligand and retention of the six-coordinate state, and is also accompanied by loss of enzyme activity. Treatment of the five-coordinate HgCl2-treated enzyme with thiols, such as homocysteine, results in reversion to a six-coordinate state. Resonance Raman spectroscopy with 34S-labeled enzyme reveals the return of the endogenous thiol ligand under these conditions and rules out direct coordination by the thiolate of homocysteine to the heme.     

Role of calcium in metalloenzymes: effects of calcium removal on the axial ligation geometry and magnetic properties of the catalytic diheme center in MauG

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and M?ssbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and M?ssbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.     

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.     

Characterization of the heme binding properties of Staphylococcus aureus IsdA

We report the first characterization of the physical and spectroscopic properties of the Staphylococcus aureus heme-binding protein IsdA. In this study, a combination of gel filtration chromatography and analytical centrifugation experiments demonstrate that IsdA, in solution, is a monomer and adopts an extended conformation that would suggest that it has the ability to protrude from the staphylococcal cell wall and interact with the extracellular environment. IsdA efficiently scavenged intracellular heme within Escherichia coli. Gel filtration chromatography and electrospray mass spectrometry together showed that rIsdA in solution is a monomer, and each monomer binds a single heme. Magnetic circular dichroism analyses demonstrate that the heme in rIsdA is a five-coordinate high-spin ferric heme molecule, proximally coordinated by a tyrosyl residue in a cavity that restricts access to small ligands. The heme binding is unlike that in a typical heme protein, for example, myoglobin, because we report that no additional axial ligation is possible in the high-spin ferric state of IsdA. However, reduction to ferrous heme is possible which then allows CO to axially ligate to the ferrous iron. Reoxidation forms the ferric heme, which is once again isolated from exogenous ligands. In summary, rIsdA binds a five-coordinate, high-spin ferric heme which is proximally coordinated by tyrosine. Reduction results in formation of five-coordinate, high-spin ferrous heme with a neutral axial ligand, most likely a histidine. Subsequent addition of CO results in a six-coordinate low-spin ferrous heme also with histidine likely bound proximally. Reoxidation returns the tyrosine as the proximal ligand.     

Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding

KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.     

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.     

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)     

Resonance Raman characterization of the heme cofactor in cystathionine beta-synthase. Identification of the Fe-S(Cys) vibration in the six-coordinate low-spin heme

Human cystathionine beta-synthase (CBS) is an essential enzyme for the removal of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is thought to be similar to that in other PLP-dependent enzymes that catalyze beta-replacement reactions, the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor of CBS in both the ferric and ferrous states using resonance Raman spectroscopy. Positive identification of a cysteine ligand was achieved by global (34)S isotopic substitution which allowed us to assign the nu(Fe-S) for the six-coordinate low-spin ferric heme at 312 cm(-1). In addition, the CO adduct of ferrous CBS has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment, and indicates that the Fe-S(Cys) bond is labile. We have also found that addition of HgCl(2) to the ferric heme causes conversion of the low-spin heme to a five-coordinate high-spin heme with loss of the cysteine ligand. The present spectroscopic studies do not support a reaction mechanism in which homocysteine binds directly to the heme via displacement of the Cys ligand in the binary enzyme complex, as had been previously proposed.     

Spectroscopic and substrate binding properties of heme-containing aldoxime dehydratases, OxdB and OxdRE

Aldoxime dehydratase (Oxd) is a novel hemeprotein that catalyzes the dehydration reaction of aldoxime to produce nitrile. In this study, we studied the spectroscopic and substrate binding properties of two Oxds, OxdB from Bacillus sp. strain OxB-1 and OxdRE from Rhodococcus sp. N-771, that show different quaternary structures and relatively low amino acid sequence identity. Electronic absorption and resonance Raman spectroscopy revealed that ferric OxdRE contained a six-coordinate low-spin heme, while ferric OxdB contained a six-coordinate high-spin heme. Both ferrous OxdRE and OxdB included a five-coordinate high-spin heme to which the substrate was bound via its nitrogen atom for the reaction to occur. Although the ferric Oxds were inactive for catalysis, the substrate was bound to the ferric heme via its oxygen atom in both OxdB and OxdRE. Electronic paramagnetic resonance (EPR) and rapid scanning spectroscopy revealed that the flexibility of the heme pocket was different between OxdB and OxdRE, which might affect their substrate specificity.     

Analysis of heme structural heterogeneity in Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a heme enzyme considered important for virulence, which is also responsible for activation of the anti-tuberculosis pro-drug isoniazid. Here, we present an analysis of heterogeneity in KatG heme structure using optical, resonance Raman, and EPR spectroscopy. Examination of ferric KatG under a variety of conditions, including enzyme in the presence of fluoride, chloride, or isoniazid, and at different stages during purification in different buffers allowed for assignment of spectral features to both five- and six-coordinate heme. Five-coordinate heme is suggested to be representative of "native" enzyme, since this species was predominant in the enzyme examined immediately after one chromatographic protocol. Quantum mechanically mixed spin heme is the most abundant form in such partially purified enzyme. Reduction and reoxidation of six-coordinate KatG or the addition of glycerol or isoniazid restored five-coordinate heme iron, consistent with displacement of a weakly bound distal water molecule. The rate of formation of KatG Compound I is not retarded by the presence of six-coordinate heme either in wild-type KatG or in a mutant (KatG[Y155S]) associated with isoniazid resistance, which contains abundant six-coordinate heme. These results reveal a number of similarities and differences between KatG and other Class I peroxidases.     

Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acid to prostaglandin H(2) (PGH(2)), the committed step in prostaglandin and thromboxane formation. Interaction of peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequent cyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopic and directed mutagenesis data with X-ray crystallographic refinement of the heme site. Optical and Raman spectroscopy of oxidized oPGHS-1 indicate that its heme iron (Fe(3+)) exists exclusively as a high-spin, six-coordinate species in the holoenzyme and in heme-reconstituted apoenzyme. The sixth ligand is most likely water. The cyanide complex of oxidized oPGHS-1 has a six-coordinate, low-spin ferric iron with a v[Fe-CN] frequency at 445 cm(-)(1); a monotonic sensitivity to cyanide isotopomers that indicates the Fe-CN adduct has a linear geometry. The ferrous iron in reduced oPGHS-1 adopts a high-spin, five-coordinate state that is converted to a six-coordinate, low-spin geometry by CO. The low-frequency Raman spectrum of reduced oPGHS-1 reveals two v[Fe-His] frequencies at 206 and 222 cm(-)(1). These vibrations, which disappear upon addition of CO, are consistent with a neutral histidine (His388) as the proximal heme ligand. The refined crystal structure shows that there is a water molecule located between His388 and Tyr504 that can hydrogen bond to both residues. However, substitution of Tyr504 with alanine yields a mutant having 46% of the peroxidase activity of native oPGHS-1, establishing that bonding of Tyr504 to this water is not critical for catalysis. Collectively, our results show that the proximal histidine ligand in oPGHS-1 is electrostatically neutral. Thus, in contrast to most other peroxidases, a strongly basic proximal ligand is not necessary for peroxidase catalysis by oPGHS-1.     

Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.     

Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

Modeling low-pH hemoproteins

A tetracoordinate ferrous heme (iron-porphyrin) has been proposed as an intermediate at low pH (less than 3.0) for respiratory hemoproteins, peroxidases, and model heme complexes. This intermediate is believed to arise via protonation of the N(epsilon) atom of the proximal histidine and consequent cleavage of the Fe-N(epsilon) bond. To establish a spectral signature for the proposed low-pH tetracoordinate species, we have obtained Soret excitation resonance Raman spectra on samples of crystallographically defined, tetracoordinate iron(II)-octaethylporphyrin (Fe.OEP; S = 1). The high-frequency (greater than or equal to 900 cm-1) resonance Raman spectral features of Fe.OEP are clearly distinct from those of high-spin pentacoordinate or low-spin hexacoordinate ferrous hemes. Rather, they are at frequencies more typically observed for low-spin hexacoordinate ferric porphyrins. Comparative spectral analysis of tetracoordinate Fe.OEP and other proposed tetracoordinate ferrous hemes (e.g. iron(II)-protoporphyrin IX) demonstrates little or no macrocycle effect on the resonance Raman frequencies above 900 cm-1. This work thus serves to provide a testable spectral signature by which the existence of the proposed tetracoordinate biological intermediate may be verified and by which its functional significance may be tested.     

Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420.

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (mos), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P420 are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal VFe-CO peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

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.