Phe393 mutants of cytochrome P450 BM3 with modified heme redox potentials have altered heme vinyl and propionate conformations

It has been well established that the heme redox potential is affected by many different factors. Among others, it is sensitive to the proximal heme ligand and the conformation of the propionate and vinyl groups. In the cytochrome P450 BM3 heme domain, substitution of the highly conserved phenylalanine 393 results in a dramatic change in the heme redox potential [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429]. We have used resonance Raman spectroscopy to characterize heme structural changes and modification of heme interactions with the protein matrix that are induced by the F393 substitutions and to determine their correlation with the heme redox potential. Our results show that the Fe-S stretching frequency of the 5-coordinated, high-spin ferric heme is not affected by the mutations, suggesting that the electron density in the Fe-S bond in this state is not affected by the F393 mutation and is not a good indicator of the heme redox potential. Substrate binding perturbs the hydrogen bonding between one propionate group and the protein matrix and correlates to both the size of residue 393 and the heme redox potential. However, heme reduction does not affect the conformation of the propionate groups. Although the conformation of the vinyl groups is not affected much by substrate binding, their conformation changes from mainly out-of-plane to predominantly in-plane upon heme reduction. The extent of these conformational changes correlates strongly with the size of the 393 residue and the heme redox potential, suggesting that steric interaction between this residue and the vinyl groups may be of importance in regulating the heme redox potential in the P450 BM3 heme domain. Further implications of our findings for the change in redox potential upon mutation of F393 will be discussed.     

Role of electrostatics and salt bridges in stabilizing the compound I radical in ascorbate peroxidase

Cytochrome c (CcP) and ascorbate peroxidase (APX) are heme peroxidases which have very similar active site structures yet differ substantially in the properties of compound I, the intermediate formed upon reaction with peroxides. Although both peroxidases have a tryptophan in the proximal heme pocket, Trp191 in CcP and Trp179 in APX, only Trp191 in CcP forms a stable cation radical while APX forms the more traditional porphyrin pi-cation radical. Previous work [Barrows, T. P., et al. (2004)Biochemistry 43, 8826-8834] has shown that converting three methionine residues in the cytochrome c peroxidase (CcP) proximal heme pocket to the corresponding residues in APX dramatically decreased the stability of the Trp191 radical in CcP compound I. On the basis of these results, we reasoned that replacing the analogous residues at positions 160, 203, and 204 in APX with methionine should stabilize a Trp179 radical in APX compound I. Steady- and transient-state kinetics of this mutant (designated APX3M) show a significant destabilization of the native porphyrin pi-radical, while electron paramagnetic resonance (EPR) studies show an increase in the intensity of the signal at g = 2.006 with characteristics consistent with formation of a Trp radical. This hypothesis was tested by replacing Trp179 with Phe in the APX3M background. The EPR spectrum of this mutant was very similar to that of the CcP W191G mutant which is known to form a tyrosine radical. Previously published theoretical studies [Guallar, V., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 6998-7002] suggest that electrostatic shielding of the heme propionates also plays a role in the stability of the porphyrin radical. Arg172 in APX hydrogen bonds with one of the heme propionates. Replacing Arg172 with an asparagine residue in the APX3M background generates a mutant which no longer forms the full complement of the compound I porphyrin pi-radical. These results suggest that the electrostatics of the proximal pocket and the shielding of propionate groups by salt bridges are critical factors controlling the location of a stable compound I radical in heme peroxidases.     

Assignment of heme resonances and determination of the electronic structures of high- and low-spin nitrophorin 2 by 1H and 13C NMR spectroscopy: an explanation of the order of heme methyl resonances in high-spin ferriheme proteins

The (1)H NMR resonances of the heme substituents of the low-spin Fe(III) form of nitrophorin 2, as its complexes with N-methylimidazole (NP2-NMeIm) and imidazole (NP2-ImH), have been assigned by a combination of (1)H homonuclear two-dimensional NMR techniques and (1)H-(13)C HMQC. Complete assignment of the proton and partial assignment of the (13)C resonances of the heme of these complexes has been achieved. Due to favorable rates of ligand exchange, it was also possible to assign part of the (1)H resonances of the high-spin heme via saturation transfer between high- and low-spin protein forms in a partially liganded NP2-NMeIm sample; additional resonances (vinyl and propionate) were assigned by NOESY techniques. The order of heme methyl resonances in the high-spin form of the protein over the temperature range of 10-37 degrees C is 8 = 5 > 1 > 3; the NMeIm complex has 5 > 1 > 3 > 8 as the order of heme methyl resonances at <30 degrees C, while above that temperature, the order is 5 > 3 > 1 > 8, due to crossover of the closely spaced 3- and 1-methyl resonances of the low-spin complex at higher temperatures. This crossover defines the nodal plane of the heme orbital used for spin delocalization as being oriented 162 +/- 2 degrees clockwise from the heme N(II)-Fe-N(IV) axis for the heme in the B orientation. For the NP2-ImH complex, the order of heme methyl resonances is 3 > 5 > 1 > 8, which defines the orientation of the nodal plane of the heme orbital used for spin delocalization as being oriented approximately 150-155 degrees clockwise from the heme N(II)-Fe-N(IV) axis. In both low-spin complexes, the results are most consistent with the exogenous planar ligand controlling the orientation of the nodal plane of the heme orbital. In the high-spin form of NP2, the proximal histidine plane is shown to be oriented 135 degrees clockwise from the heme N(II)-Fe-N(IV) axis, again for the B heme orientation. A correlation between the order of heme methyl resonances in the high-spin form of NP2 and several other ferriheme proteins and an apparent 90 degrees shift in the nodal plane of the orbital involved in spin delocalization from that expected on the basis of the orientation of the axial histidine imidazole nodal plane have been explained in terms of bonding interactions between Fe(III), the axial histidine imidazole nitrogen, and the porphyrin pi orbitals of the high-spin protein.     

Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.     

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.     

Probing heme propionate involvement in transmembrane proton transfer coupled to electron transfer in dihemic quinol:fumarate reductase by 13C-labeling and FTIR difference spectroscopy

Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer.     

Dynamics comparison of two myoglobins with a distinct heme active site

Sperm whale myoglobin (swMb) is a well-studied heme protein, both experimentally and theoretically. Comparatively, little attention has been paid to another member of Mb family, Aplysia limacina myoglobin (apMb). swMb and apMb have the same overall structure and perform the same biological function, i.e., O₂ carrier, while using a distinct heme active site. To provide insights into the structure-function relationship for these two Mbs, we herein made a comparison in terms of their dynamics properties using molecular dynamics simulation. We analyzed the overall structure and protein motions, as well as the intramolecular contacts, namely salt-bridges and hydrogen bonds, especially the interactions between the heme propionate groups and the polypeptide chain. The internal cavities in apMb were also compared to the well-known xenon and other cavities in swMb. Based on current simulations, we propose a unique “arginine gate” for apMb, which has a similar function to the histidine gate observed for swMb in previous studies. This study provides valuable information for understanding the homology of heme proteins, and also aids in rational design of structural and functional heme proteins by alternating the heme active site.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

High pressure, a tool for exploring heme protein active sites

High pressure is an interesting and suitable parameter in the study of the dynamics and stability of proteins. The effects of pressure on proteins delineates its volumic (deltaV degrees ) and energetic (deltaG degrees ) parameters. An enormous amount of effort has been invested by several laboratories in developing basic theory and high pressure techniques that allow the determination of barotropic parameters. Cytochrome P450s, one of the largest super families of heme proteins, are good models for high pressure studies. Two distinct pressure-induced spin transitions of the heme iron in the active site and a P450 to P420 inactivation process have been characterized. The obtained reaction volumes of these two processes for a series of analog-bound cytochrome P450s are compared. We have shown that both the spin volume and the inactivation volume are dependent on the substrate analogs which are known to modulate the polarity and hydration of the heme pocket. Several linear correlations were found between these reaction volumes and the physico-chemical properties of the heme protein such as the polarity-induced exposure of tyrosines, the hydration of the cytochrome CYP101 heme pocket, and the mobility and binding of the substrates indicate that they constitute the main contribution to the complex thermodynamic reaction volume parameters. This interpretation allows us to conclude that cytochrome CYP101, CYP2B4 and CYP102 possess a similar mechanism of substrate binding. Interestingly the barotropic behaviors of monomeric cytochrome P450s are quite different from those of oligomeric and hetorooligomeric cytochrome P450s. The interactions of heterooligomeric subunits influence the stability of individual cytochrome P450s and the asymmetric organization of subunits which can control and modulate the activity and the recognition with NADPH-cytochrome P450 reductase.     

The mechanism of heme transfer from the cytoplasmic heme binding protein PhuS to the delta-regioselective heme oxygenase of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa has evolved two outer membrane receptor-mediated uptake systems (encoded by the phu and has operons) by which it can utilize the hosts heme and hemeproteins as a source of iron. PhuS is a cytoplasmic heme binding protein encoded within the phu operon and has previously been shown to function in the trafficking of heme to the iron-regulated heme oxygenase (pa-HO). While the heme association rate for PhuS was similar to that of myoglobin, a markedly higher rate of heme dissociation (approximately 10(5) s(-1)) was observed, in keeping with a function in heme-trafficking. Additionally, the transfer of heme from PhuS to pa-HO was shown to be specific and unidirectional when compared to transfer to the non-iron regulated heme oxygenase (BphO), in which heme distribution between the two proteins merely reflects their relative intrinsic affinities for heme. Furthermore, the rate of transfer of heme from holo-PhuS to pa-HO of 0.11 +/- 0.01 s(-1) is 30-fold faster than that to apo-myoglobin, despite the significant higher binding affinity of apo-myoglobin for heme (kH = 1.3 x 10(-8) microM) than that of PhuS (0.2 microM). This data suggests that heme transfer to pa-HO is independent of heme affinity and is consistent with temperature dependence studies which indicate the reaction is driven by a negative entropic contribution, typical of an ordered transition state, and supports the notion that heme transfer from PhuS to pa-HO is mediated via a specific protein-protein interaction. In addition, pH studies, and reactions conducted in the presence of cyanide, suggest the involvement of spin transition during the heme transfer process, whereby the heme undergoes spin change from 6-c LS to 6-c HS either in PhuS or pa-HO. On the basis of the magnitudes of the activation parameters obtained in the presence of cyanide, whereby both complexes are maintained in a 6-c LS state, and the biphasic kinetics of heme transfer from holo-PhuS to pa-HO-wt, supports the notion that the spin-state crossover occur within holo-PhuS prior to the heme transfer step. Alternatively, the lack of the biphasic kinetic with pa-HO-G125V, 6-c LS, and with comparable rate of heme transfer as pa-HO is supportive of a mechanism in which the spin-change could occur within pa-HO. The present data suggests either or both of the two pathways proposed for heme transfer may occur under the present experimental conditions. The dissection of which pathway is physiologically relevant is the focus of ongoing studies.     

NMR investigation of the heme electronic structure in deoxymyoglobin possessing a fluorinated heme

The heme electronic structures of deoxymyoglobins (deoxy-Mbs) reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-(trifluoromethyl)porphyrinatoiron(III) (7-PF), 13,17-bis(2-carboxylatoethyl)-3,7-difluoro-2,8,12,18-tetramethylporphyrinatoiron(III) (3,7-DF), and 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethylporphyrinatoiron(III) (2-MF) have been characterized by ¹H and ¹⁹F NMR. The analysis of heme methyl proton shift patterns of the hemes in their bis-cyano forms demonstrated that, owing to the substitution of a strongly electron-withdrawing perfluoromethyl group, CF₃, to porphyrin, the porphyrin -system of 7-PF is more significantly distorted from four-fold symmetry than those of the ring-fluorinated hemes, 3,7-DF and 2-MF. The presence of the heme orientation disorder resulted in the observation of the two well-resolved ¹⁹F signals in the spectra of deoxy-Mbs possessing 7-PF and 2-MF. The ¹⁹F signals of deoxy-Mb possessing 7-PF exhibited a relatively large difference in paramagnetic shift (~30 ppm), despite their small paramagnetic shifts (~30 ppm), supporting the significant contribution of a spin delocalization mechanism in this Mb due to the d-electron configuration derived from the ⁵E ground state. On the other hand, ¹⁹F signals of deoxy-Mbs with 3,7-DF as well as 2-MF exhibited large paramagnetic shifts (~250 ppm) with a relatively small difference in the paramagnetic shift (~20 ppm), indicating the predominant contribution of spin delocalization, due to a d-electron configuration derived from the ⁵B₂ ground state. These results demonstrate for the first time that the relative contributions of the orbital ground states derived from ⁵E and ⁵B₂ states to the heme electronic structure in deoxy-Mb are affected by the distortion of the porphyrin -system exerted by chemical properties of the heme peripheral side-chains.Abbreviations 3,7-DF 13,17-bis(2-carboxylatoethyl)-3,7-difluoro-2,8,12,18-tetramethylporphyrinatoiron(III) - 2-MF 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethylporphyrinatoiron(III) - 7-PF 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-(trifluoromethyl)porphyrinatoiron(III) - Mb myoglobin - Mb(7-PF) deoxy-Mb reconstituted with 7-PF - Mb(3,7-DF) deoxy-Mb reconstituted with 3,7-DF - Mb(2-MF) deoxy-Mb reconstituted with 2-MF - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy     

Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand

In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows: -481 mV (WT), -477 mV (A320Q), -453 mV (F310A), and -450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H2O2 show the following trend: WT approximately equal to A320Q < F310A approximately equal to F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H2O2.     

Crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate. Conformational change of the distal helix during the heme cleavage reaction

The crystal structure of rat heme oxygenase-1 in complex with biliverdin-iron chelate (biliverdin(Fe)-HO-1), the immediate precursor of the final product, biliverdin, has been determined at a 2.4-A resolution. The electron density in the heme pocket clearly showed that the tetrapyrrole ring of heme is cleaved at the alpha-meso edge. Like the heme bound to HO-1, biliverdin-iron chelate is located between the distal and proximal helices, but its accommodation state seems to be less stable in light of the disordering of the solvent-exposed propionate and vinyl groups. The middle of the distal helix is shifted away from the center of the active site in biliverdin(Fe)-HO-1, increasing the size of the heme pocket. The hydrogen-bonding interaction between Glu-29 and Gln-38, considered to restrain the orientation of the proximal helix in the heme-HO-1 complex, was lost in biliverdin(Fe)-HO-1, leading to relaxation of the helix. Biliverdin has a distorted helical conformation; the lactam oxygen atom of its pyrrole ring-A interacted with Asp-140 through a hydrogen-bonding solvent network. Because of the absence of a distal water ligand, the iron atom is five-coordinated with His-25 and four pyrrole nitrogen atoms. The coordination geometry deviates considerably from a square pyramid, suggesting that the iron may be readily dissociated. We speculate that the opened conformation of the heme pocket facilitates sequential product release, first iron then biliverdin, and that because of biliverdin's increased flexibility, iron release triggers its slow dissociation.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).     

1H-NMR study of heme propanoate mobility in the active site of myoglobin from Galeorhinus japonicus

Time-dependent NOE studies of the C13(1) and C17(1) methylene proton resonances of the heme peripheral propanoate groups have elucidated their mobility in the active site of the ferric high-spin form of Galeorhinus japonicus myoglobin. A large difference in the chemical shift due to the non-equivalence of the heme C13(1) and C17(1) methylene proton resonances allows selective irradiation of a given proton resonance by a high-power selective decoupler pulse in spite of their fast relaxation rates. NOE accumulation of the resonance of one methylene proton after saturation of the resonance of the other proton essentially follows the theoretical prediction derived using the two-spin approximation, and the cross-relaxation rates for the heme C13(1) and C17(1) methylene proton spin systems were quantitatively determined. The correlation time for the mobility of the internuclear vector connecting the heme C13(1) or C17(1) methylene protons was then calculated from the cross-relaxation rate and values of approximately 11 ns were obtained for both C13(1) and C17(1) methylene groups in 2 mM Galeorhinus japonicus myoglobin at 35 degrees C. The immobile C13(1) and C17(1) methylenes of the heme propanoate groups, together with a large difference in chemical shift between the methylene proton resonances, dictate their fixed orientation with respect to the protein moiety as well as the heme plane, and are therefore consistent with the immobile heme in the active site of myoglobin.     

The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.

Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.     

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase.

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].     

Novel X-ray sequences and crystal structures of Persian and Starry sturgeon methemoglobins: Highlighting the role of heme pocket waters in causing autoxidation

Although there is a high sequence similarity between mammalian and fish hemoglobin (Hb), the oxidation and heme loss rates can vary greatly between them such that fish Hbs oxidise much more rapidly than mammalian Hbs. There is to date no sequence or structural data for any sturgeon Hb to reveal the level of autoxidation in these fish. In this study, novel high resolution X-ray sequences and crystal structures of methemoglobin (Met-Hb) from two sturgeon fish including Persian sturgeon (Acipenser percisus) and Starry sturgeon (Acipenser stellatus) belonging to the Caspian sea has been determined. A comprehensive sequence and structure comparison between these sturgeon Met-Hbs and a number of non-sturgeon and normal and sickle cell anaemia human Hb in varying heme states has been carried out highlighting (i) the structural variability in the heme propionate groups; (ii) the existence of certain residues or their displacement and shift in the heme pocket allowing entry of water molecules into the heme pocket; (iii) the importance of the number of water molecules in the heme pocket; (iv) the hydrogen bonding between oxygens of A and D propionate groups and that of waters in the heme pocket; and (v) the role of heme binding waters causing oxidative stress and heme autoxidation.     

Identification of heme propionate vibrational modes in the resonance Raman spectra of cytochrome c oxidase

The propionate groups of heme a and a₃ in cytochrome c oxidase (CcO) have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a₃, as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H₂O–D₂O isotope substitution experiments combined with wavelength-selective resonance enhancement (for bCcO) or mutagenesis studies (for RsCcO).