Amphitrite ornata dehaloperoxidase (DHP): investigations of structural factors that influence the mechanism of halophenol dehalogenation using "peroxidase-like" myoglobin mutants and "myoglobin-like" DHP mutants

Dehaloperoxidase (DHP), discovered in the marine terebellid polychaete Amphitrite ornata, is the first heme-containing globin with a peroxidase activity. The sequence and crystal structure of DHP argue that it evolved from an ancient O(2) transport and storage globin. Thus, DHP retains an oxygen carrier function but also has the ability to degrade halophenol toxicants in its living environment. Sperm whale myoglobin (Mb) in the ferric state has a peroxidase activity ～10 times lower than that of DHP. The catalytic activity enhancement observed in DHP appears to have been generated mainly by subtle changes in the positions of the proximal and distal histidine residues that appeared during DHP evolution. Herein, we report investigations into the mechanism of action of DHP derived from examination of "peroxidase-like" Mb mutants and "Mb-like" DHP mutants. The dehalogenation ability of wild-type Mb is augmented in the peroxidase-like Mb mutants (F43H/H64L, G65T, and G65I Mb) but attenuated in the Mb-like T56G DHP variant. X-ray crystallographic data show that the distal His residues in G65T Mb and G65I are positioned ～0.3 and ～0.8 ?, respectively, farther from the heme iron compared to that in the wild-type protein. The H93K/T95H double mutant Mb with the proximal His shifted to the "DHP-like" position has an increased peroxidase activity. In addition, a better dehaloperoxidase (M86E DHP) was generated by introducing a negative charge near His89 to enhance the imidazolate character of the proximal His. Finally, only minimal differences in dehalogenation activities are seen among the exogenous ligand-free DHP, the acetate-bound DHP, and the distal site blocker L100F DHP mutant. Thus, we conclude that binding of halophenols in the internal binding site (i.e., distal cavity) is not essential for catalysis. This work provides a foundation for a new structure-function paradigm for peroxidases and for the molecular evolution of the dual-function enzyme DHP.     

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.     

Resonance Raman study of ferric heme adducts of dehaloperoxidase from Amphitrite ornata

The study of axial ligation by anionic ligands to ferric heme iron by resonance Raman spectroscopy provides a basis for comparison of the intrinsic electron donor ability of the proximal histidine in horse heart myoglobin (HHMb), dehaloperoxidase (DHP), and horseradish peroxidase (HRP). DHP is a dimeric hemoglobin (Hb) originally isolated from the terebellid polychaete Amphitrite ornata. The monomers are structurally related to Mb and yet DHP has a peroxidase function. The core size marker modes, v2 and v3, were observed using Soret excitation, and DHP-X was compared to HHMb-X for the ligand series X = F, Cl, Br, SCN, OH, N3, and CN. Special attention was paid to the hydroxide adduct, which is also formed during the catalytic cycle of peroxidases. The Fe-OH stretching frequency was observed and confirmed by deuteration and is higher in DHP than in HHMb. The population of high-spin states of the heme iron in DHP was determined to be intermediate between HHMb and HRP. The data provide the first direct measurement of the effect of axial ligation on the heme iron in DHP. The Raman data support a modified charge relay in DHP, in which a strongly hydrogen-bonded backbone carbonyl (>C=O) polarizes the proximal histidine. The charge relay mechanism by backbone carbonyl >C=O-His-Fe is the analogue of the Asp-His-Fe of peroxidases and Glu-His-Fe of flavohemoglobins.     

EPR and ENDOR studies of cryoreduced compounds II of peroxidases and myoglobin. Proton-coupled electron transfer and protonation status of ferryl hemes

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.     

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)     

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.     

Enzyme function of the globin dehaloperoxidase from Amphitrite ornata is activated by substrate binding

Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.     

Degradation of sulfide by dehaloperoxidase-hemoglobin from <Emphasis Type="Italic">Amphitrite ornata</Emphasis>

Dehaloperoxidase-hemoglobin (DHP) is a unique multifunctional enzyme with a globin fold. The enzyme serves as the respiratory hemoglobin for the marine worm Amphitrite ornata and has been shown to catalyze the conversion of highly toxic trihalophenols to dihaloquinones as a detoxification function for the organism. Given the simplicity of the structure of A. ornata, it is entirely possible that DHP may play an even more general role in detoxification of the organism from sulfide commonly found in the coastal estuaries where A. ornata thrives. Comparison of DHP with other sulfide-binding hemoglobins shows that DHP possesses several distal cavity structural properties, such as an aromatic cage and a hydrogen-bond-donor amino acid (His55), that facilitate sulfide binding. Furthermore, a complete reduction of the ferric heme occurs after sulfide exposure under aerobic or anaerobic conditions to yield either the oxy or the deoxy ferrous states of DHP, respectively. Oxidation of sulfide by the heme leads to sulfur products that are less toxic to A. ornata. This proposed new function for DHP relies on the highly flexible distal His55 for deprotonation of the bound hydrogen sulfide, similar to H₂O₂ activation of the peroxidase function, and provides further support for the importance of the flexibility of the distal His55 in this novel globin.     

Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.     

Flavohemoglobin, a globin with a peroxidase-like catalytic site

Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry.     

Structural basis of human cytoglobin for ligand binding

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.     

Functional consequences of the creation of an Asp-His-Fe triad in a 3/3 globin

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.     

NMR study of the active site of resting state and cyanide-inhibited lignin peroxidase from Phanerochaete chrysosporium. Comparison with horseradish peroxidase

One- and two-dimensional 1H NMR spectroscopy has been used to probe the active site of the high spin ferric resting state and the low spin, cyanide-inhibited derivative of isozyme H2 of the lignin peroxidase, LiP, from Phanerochaete chrysosporium strain BKM 1767. One-dimensional NMR revealed a resting state LiP that is five coordinate at 25 degrees C with an electronic structure similar to that of horseradish peroxidase, HRP. Differential paramagnetic relaxivity was used to identify the C beta H signals of the axial His177. A combination of bond correlation spectroscopy and nuclear Overhauser effect spectroscopy of cyanide-inhibited LiP (LiP-CN) has allowed the assignment of all resolved heme resonances without recourse to isotope labeling, as well as those of the proximal His177 and the distal His48. The surprising effectiveness of the two dimensional NMR methods on such a large and paramagnetic protein indicates that such two dimensional experiments can be expected to have major impact on solution structure determination of diverse classes of heme peroxidases. The two dimensional NMR data of LiP-CN reveal a heme contact shift pattern that reflects a close similarity to that of HRP-CN, including the unusual in-plane trans and cis orientation of the 2- and 4-vinyls. The axial His177 also exhibits the same orientation relative to the heme as in HRP-CN. The proximal His177 contact shifted resonances of both the low spin LiP-CN and high spin LiP are shown to reflect significantly reduced hydrogen bond donation by, or imidazolate character for, the axial histidine in LiP relative to HRP, which may explain the higher redox potential of LiP. The signals are identified for a distal residue that originates from the protonated His48 with disposition relative to the heme similar to that found for the distal His42 in HRP-CN. In contrast, the absence of any resolved signals attributable to an Arg44 in LiP-CN suggest that this distal residue has an altered orientation relative to the heme compared with that of the conserved Arg38 in HRP-CN (Thanabal, V., de Ropp, J. S., and La Mar, G. N. (1987) J. Am. Chem. Soc. 109, 7516-7525).     

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.     

Structural and dynamic properties of Synechocystis sp. PCC 6803 Hb revealed by reconstitution with Zn-protoporphyrin IX

In its resting state, the truncated globin of the cyanobacterium Synechocystis sp. PCC 6803 exhibits hexacoordination of the heme iron, with His46 (E10) and His70 (F8, proximal) serving as axial ligands. Diatomic ligands displace the distal His46 (E10) from the ferric and ferrous iron and promote considerable structural changes in the B helix, E helix, and EF regions. Here, Zn(II)-substituted hemoglobin was used to explore the role of distal ligands in stabilizing the heme pocket structure. NMR data showed that the Zn ion was coordinated by the four pyrrole nitrogens and by His70 (F8) only. The proximal side of the Zn-porphyrin adopted a geometry recognizable as that of the wild-type protein. Decoordination of His46 (E10) to form the pentacoordinate Zn resulted in an incomplete transition to the conformation observed in the ferric, cyanide-bound protein. The NMR data also demonstrated that the H helix underwent complex dynamic processes near His117, a residue readily reacting with the wild-type heme 2-vinyl group in a post-translational modification.     

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants

In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.     

Proton magnetic resonance studies of peroxidases from turnip and horseradish.

Proton NMR spectra at 270 MHz have been measured for horseradish peroxidase and turnip peroxidase isoenzymes (P1, P2, P3 and P7) in both their high spin ferric native states and as the low spin ferric cyanide complexes. Resonances of amino acids near the heme have been identified and used to investigate variations in the structure of the heme crevice amongst the enzymes. Ligand proton resonances have been resolved in spectra of the cyanide complexes of the peroxidases and these provide information on the heme electronic structure. The electronic structure of the heme and the tertiary structure of the heme crevice are essentially the same in the acidic turnip isoenzymes, P1, P2 and, to a lesser extent, P3 but differ in the basic turnip enzyme, P7. The heme electronic structure and nature of the iron ligands in peroxidases are discussed. Further evidence is presented for histidine as the proximal ligand. A heme-linked ionizable group with a pK of 6.5 has been detected by NMR in the cyanide complex of horseradish peroxidase.     

Presence of endogenous calcium ion and its functional and structural regulation in horseradish peroxidase

The endogenous calcium ion (Ca2+) in horseradish peroxidase (HRP) was removed to cause substantial changes in the proton NMR spectra of the enzyme in various oxidation/spin states. The spectral changes were interpreted as arising from the substantial alterations in the heme environments, most likely the heme proximal and distal sides. The comparative kinetic and redox studies revealed that these conformational changes affect the reduction process of compound II, resulting in the decrease of the enzymatic activity of HRP. It is also revealed from the ESR spectrum and the temperature dependences of the NMR and optical absorption spectra of the Ca2+-free enzyme that the heme iron atom of the Ca2+-free enzyme is in a thermal spin mixing between ferric high and low spin states, in contrast to that of the native enzyme. These results show that Ca2+ functions in maintaining the protein structure in the heme environments as well as the spin state of the heme iron, in favor of the enzymatic activity of HRP.     

Preparation and spectral characterization of the heme d1.apomyoglobin complex: an unusual protein environment for the substrate-binding heme of Pseudomonas cytochrome oxidase

The heme d1 prosthetic group isolated from Pseudomonas cytochrome oxidase combines with apomyoglobin to form a stable, optically well-defined complex. Addition of ferric heme d1 quenches apomyoglobin tryptophan fluorescence suggesting association in a 1:1 molar ratio. Optical absorption maxima for heme d1.apomyoglobin are at 629 and 429 nm before, and 632 and 458 nm after dithionite reduction; they are distinct from those of heme d1 in aqueous solution but more similar to those unobscured by heme c in Pseudomonas cytochrome oxidase. Cyanide, carbon monoxide and imidazole alter the spectrum of heme d1.apomyoglobin demonstrating axial coordination to heme d1 by exogeneous ligands. The cyanide-induced optical difference spectra exhibit isosbestic points, and a Scatchard-like analysis yields a linear plot with an apparent dissociation constant of 4.2 X 10(-5) M. However, carbon monoxide induces two absorption spectra with Soret maxima at 454 or 467 nm, and this duplicity, along with a shoulder that correlates with the latter before binding, suggests multiple carbon monoxide and possibly heme d1 orientations within the globin. The 50-fold reduction in cyanide affinity over myoglobin is more consistent with altered heme pocket interactions than the intrinsic electronic differences between the two hemes. However, stability of the heme d1.apomyoglobin complex is verified further by the inability to separate heme d1 from globin during dialysis and column chromatography in excess cyanide or imidazole. This stability, together with a comparison between spectra of ligand-free and -bound derivatives of heme d1-apomyoglobin and heme d1 in solution, implies that the prosthetic group is coordinated in the heme pocket through a protein-donated, strong-field ligand. Furthermore, the visible spectrum of heme d1.apomyoglobin varies minimally with ligand exchange, in contrast to the Soret, which suggests that much spectral information concerning heme d1 coordination in the oxidase is lost by interference from heme c absorption bands. A comparison of the absorption spectra of heme d1.apomyoglobin and Pseudomonas cytochrome oxidase, together with a critical examination of the previous axial ligand assignments from magnetic resonance techniques in the latter, implies that it is premature to accept the assignment of bishistidine heme d1 coordination in oxidized, ligand-free oxidase and other iron-isobacteriochlorin-containing enzymes.     

Thermochromism of heme adducts of Glycera hemoglobin and some other monomeric heme proteins

The thermally induced difference spectra of myoglobin (Mb) and Glycera dibranchiata hemoglobin (Hbm) derivatives and of cytochrome-c were recorded between 4 degrees and 30 degrees C in the 390-750 nm range. Thermodynamic parameters were estimated and upper and lower temperature limiting spectra were deduced for the various heme protein derivatives' equilibria. The effective iron d-electron population divides the hemes broadly into two different groups of behavior type. In the first group, Hbm(III)N3, Hbm(III), Mb(III)(H2O), and Cytc(III) show equilibria between two spin states. The weakest coupling between the heme and the globin occurs among the second group, for Hbm(II)CO and Mb(II)CO, which in the higher temperature limit undergoes averaging of the carbonyl tilt, while an axially elongated geometry is probably accessed for Hbm(II)NO and Mb(II)NO. Examples of the less common situation of increased absorption intensity and/or low-spin states at higher temperature were found in both groups. In the case of the methyl thioglycolate low-spin adducts of Hbm(III), an acid/base equilibrium involving thioglycolate deprotonation occurs. Apparent enthalpy-entropy compensation is exhibited by all these heme derivatives, and it is suggested that the delta H degrees and delta S degrees values relate to the intimacy of coupling between the heme structure and the solvent-dependent microconformation of the globin.