Heme Active Site Research Articles

Role of the lateral channel in catalase HPII of Escherichia coli.

The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.

Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T).

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.

Chiral Recognition at the Heme Active Site of Nitric Oxide Synthase Is Markedly Enhanced byl-Arginine and 5,6,7,8-Tetrahydrobiopterin

The effects of substrate,l-Arg and cofactors, (6R)-l-erythro-5, 6, 7, 8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for theS-andR-enantiomers of ligand I (S/R) was 30, while theS/Rratio for ligand II and theR/Sratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 μM H4B. However, in the presence of 1 mMl-Arg, theS/Rratio of theKdvalues for ligand I was decreased down to 5.9. In the presence of both 1 mMl-Arg and 0.1 mM H4B, theS/Rratios for ligands I and II and theR/Sratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.

The Crucial Roles of Asp-314 and Thr-315 in the Catalytic Activation of Molecular Oxygen by Neuronal Nitric-oxide Synthase: A SITE-DIRECTED MUTAGENESIS STUDY

Nitric-oxide synthase (NOS) is a flavohemoprotein that has a cytochrome P450 (P450)-type heme active site and catalyzes the monooxygenation of L-Arg to NG-hydroxy-L-Arg (NHA) according to the normal P450-type reaction in the first step of NO synthesis. However, there is some controversy as to how the second step of the reaction, from NHA to NO and L-citrulline, occurs within the P450 domain of NOS. By referring to the heme active site of P450, it is conjectured that polar amino acid(s) such as Asp/Glu and Thr must be responsible for the activation of molecular oxygen in NOS. In this study, we have created Asp-314-->Ala and Thr-315-->Ala mutants of neuronal NOS, both of which had absorption maxima at 450 nm in the spectra of the CO-reduced complexes and studied NO formation rates and other kinetic parameters as well as the substrate binding affinity. The Asp-314-->Ala mutant totally abolished NO formation activity and markedly increased the rate of H2O2 formation by 20-fold compared with the wild type when L-Arg was used as the substrate. The NADPH oxidation and O2 consumption rates for the Asp-314-->Ala mutant were 60-65% smaller than for the wild type. The Thr-315-->Ala mutant, on the other hand, retained NO formation activity that was 23% higher than the wild type, but like the Asp-314-->Ala mutation, markedly increased the H2O2 formation rate. The NADPH oxidation and O2 consumption rates for the Thr-315-->Ala mutant were, respectively, 56 and 27% higher than for the wild type. When NHA was used as the substrate, similar values were obtained. Thus, we propose that Asp-314 is crucial for catalysis, perhaps through involvement in the stabilization of an oxygen-bound intermediate. An important role for Thr-315 in the catalysis is also suggested.

A 1H NMR study of structurally relevant inter-segmental hydrogen bond in cytochrome c

NMR signal arising from His 26 N ε H proton in horse and tuna ferrocytochromes c has been assigned. This His residue is highly conserved in most mitochondorial cytochromes c and X-ray crystallographic studies strongly suggested that its side-chain imidazole participates in an internal hydrogen bond network which is relevant to the stability of the non-helical protein folding near the heme active site. The shift and line width of the assigned signal indicated that this NH hydrogen is indeed involved in an internal hydrogen bond. On the basis of the X-ray crystal structures, the carbonyl oxygen of the residue at 44 is thought to act as a proton-acceptor for this hydrogen. The observation of nuclear Overhauser effect correlation between His 26 C ε H and Asn 31 main-chain amide NH proton signals in the present proteins also demonstrated the formation of the hydrogen bond between these residues. Consequently, the presence of a unique triad hydrogen bond network in these cytochromes c in solution has been confirmed. Taking advantage of the sensitivity of His 26 N ε H proton signal to the structural properties of this hydrogen bond network, influences of the presence of high concentration of salt or various concentrations of denaturant on the protein folding were inferred from the analysis of the NMR spectral parameters of the signal.

EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and tyrosyl radical intermediates.

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pKa value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C2,6 and C3,5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor.

Relationship between the heme active site of Arthromyces ramosus peroxidase and its oxidation activity for luminol

Relationship between the heme active site of Arthromyces ramosus peroxidase and its oxidation activity for luminol

Functional interactions in cytochrome P450BM3: flavin semiquinone intermediates, role of NADP(H), and mechanism of electron transfer by the flavoprotein domain.

Cytochrome P450BM3 is a self-sufficient soluble fatty acid hydroxylase from Bacillus megaterium utilizing tightly bound FAD and FMN cofactors to transfer reducing equivalents from NADPH to the heme active site. Active-inactive transitions of cytochrome P450BM3 were exploited to identify catalytic intermediates of the enzyme. Shortly upon reduction by NADPH, a two-electron reduced active P450BM3 is formed with two flavin semiquinones, anionic and neutral, present simultaneously. P450BM3 inactivated by NADPH has a three-electron reduced flavoprotein domain. NADPH is unable to reduce P450BM3 rapidly unless the flavoprotein domain is fully oxidized. During steady-state hydroxylation of a poor substrate, tetradecanol, the flavoprotein reduction state does not exceed two, with two flavin semiquinones, anionic and neutral, present. Absorbance and EPR spectroscopic characterization of both anionic and neutral flavin semiquinone is presented. NADPH and NADH were compared as electron donors for P450BM3-catalyzed fatty acid hydroxylation and cytochrome c and heme iron reduction. The Km for NADH of 3-5 mM is about 3000 times higher than the Km of 1-1.5 microM for NADPH. Although NADH can support cytochrome c reduction and fatty acid hydroxylation with the rates as high as 22 and 13 s-1, respectively, these turnover numbers are only about 20% of those observed with NADPH. The results suggest that nucleotide binding plays an important role in catalysis by controlling electron-transfer properties of the flavin cofactors. In W574G and G570D mutant P450BM3 enzymes that are deficient in FMN, NADP+ binding stabilizes fully reduced FAD. P450BM3 catalyzes single-turnover and steady-state laurate hydroxylation with near stoichiometric product formation at NADPH concentrations below that of the enzyme. A mechanism of electron transfer by the flavoprotein domain of P450BM3 is proposed with the reduction state of the flavoprotein domain cycling in a 0-2-1-0 sequence. We also propose that an interaction of bound NADP+ with anionic FAD semiquinone is essential for splitting a pair of electrons that are then transferred in two one-electron transfer steps to the heme catalytic site.

Transient Raman Observations of Heme Electronic and Vibrational Photodynamics in Deoxyhemoglobin

Transient resonance Raman spectroscopy has been used to probe the vibrational dynamics of the heme active site of deoxyhemoglobin during photoexcitation. Near UV pulses of approximately 35 ps in duration were used to both excite the sample and generate resonance Raman spectra of the heme during its rapid electronic and vibrational relaxation. The behavior of the Stokes and anti-Stokes transitions as a function of incident laser flux directly reflects net heme vibrational populations and permits the isolation and characterization of ground and excited electronic state phenomena. Scattering from excited electronic states significantly influences the spectra only at the highest excitation fluxes used in this study. A simple model that accounts for the flux-dependent manifestations of the electronic and vibrational contributions to the heme transient resonance Raman spectra is discussed. In addition, the data presented here clearly show mode selectivity in the vibrational energy distribution associated with t...

A nuclear paramagnetic relaxation study of the interaction of the cyclopentanedione substrate with chloroperoxidase

Association of the physiological substrate cyclopentanedione (CPDO) with chloroperoxidase (CPO) was confirmed from both line-width and longitudinal relaxation time measurements of the proton NMR signals of CPDO. A dissociation constant of 33 mM for the enzyme-substrate complex was calculated from proton NMR relaxation experiments. Chloride ion does not affect the stability of the complex, but iodide ion greatly decreased the stability of the CPO-CPDO complex. Binding of cyanide ion to the heme iron center of CPO also decreased the stability of the CPO-CPDO complex. This result indicates the critical influence of perturbation in the heme active site on the affinity of CPO toward its substrate. Titration of CPO with CPDO revealed that CPDO does not bind directly to the heme iron of CPO. A distance of 7.1 Å between the heme center of CPO and the protons of bound CPDO is deduced from the Solomon-Bloembergen relationship. This distance is 1 to 3 Å shorter than those reported for the complexes formed between CPO and organic sulfide and unreactive phenol substrates, but the distance approximates that for reactive phenol substrates.

Interactions between substrate analogues and heme ligands in nitric oxide synthase.

The substrate binding site in nitric oxide synthase (NOS) can accommodate the physiological substrates, L-arginine and N(omega)-hydroxy L-arginine as well as many substrate analogues and inhibitors. Resonance Raman spectra of carbon monoxide-bound NOS were measured to determine how these substrates and analogues interact with heme, the prosthetic group which activates oxygen for the catalytic generation of NO and citrulline from arginine in the enzyme. Two distinct conformations of the Fe-C-O moiety were detected in the resonance Raman spectra, although in the optical absorption spectra the two species are indistinguishable. In one, termed the beta-form, the Fe-CO stretching frequency and the C-O stretching frequency, located at approximately 487 and approximately 1949 cm(-1), respectively, demonstrate that the Fe-C-O group adopts a linear conformation perpendicular to the heme plane ("open" structure). In the other, termed the alpha-form, frequencies of approximately 502 and approximately 1929 cm(-1), respectively, indicate that the binding properties of bound CO are significantly affected by its immediate environment thereby leading to a "closed" structure. In the presence of L-arginine or N(omega)-OH-L-arginine all of the molecules exhibit the closed structure, indicating that the substrates exert a strong polar (and/or steric) effect on the heme-bound ligand. In the absence of any substrate or inhibitor only half of the heme population adopts the open structure whereas the rest of the heme content retains the closed conformation. It is proposed that in this population with the closed structure tetrahydrobiopterin, a cofactor of NO synthase, may reside in close proximity to the heme-bound ligand and interact with it in a similar manner as do substrates. The inverse correlation between the Fe-CO and C-O stretching modes suggests that in NOS the bonding of the cysteine to the heme iron may be weaker, as found in chloroperoxidase, than in cytochrome P-450 enzymes. This work continually proves resonance Raman spectroscopy as a powerful probe for the interactions between substrate/inhibitor and the heme active site of proteins.

X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH

Myeloperoxidase (MPO), an important enzyme in the oxygen-dependent host defense system of human polymorphonuclear leukocytes, utilizes hydrogen peroxide to catalyze the production of hypochlorous acid, an oxidizing bactericidal agent. While MPO shows significant sequence homology with other peroxidases and this homology is particularly striking among the active-site residues, MPO exhibits unusual spectral features and the unique ability to catalyze the oxidation of chloride ions. We have investigated the MPO active-site with X-ray absorption (XAS) and resonance Raman (RRS) spectroscopies at neutral pH and also at the physiological acidic pH (pH∼3) and have compared these results with those of horseradish peroxidase (HRP). At pH 7.5, XAS results show that the iron heme active site is 6-coordinate where the distal ligand is likely nitrogen or oxygen, but not sulfur. The heme is distorted compared to HRP, other peroxidases, and heme compounds, but at pH∼3, the distal ligand is lost and the heme is less distorted. RRS results under identical pH conditions show that the skeletal core-size sensitive modes and ν 3 are shifted to higher frequency at pH∼3 indicating a 6- to 5-coordination change of high spin ferric heme. In addition, a new band at 270 cm −1 is observed at pH∼3 which is consistent with the loss of the sixth ligand. The higher symmetry of the heme at pH∼3 is reflected by a single ν 4 mode in the (RRS) spectrum. HRP also loses its loosely associated distal water at this pH, but little change in heme distortion is observed. This change suggests that loss of the distal ligand in MPO releases stress on the heme which may facilitate binding of chloride ion.

Paramagnetic NMR Spectroscopy of Microperoxidase-8

Microperoxidase-8 (MP8) is the heme octapeptide derived from enzymatic proteolysis of horse-heart cytochrome c. Not only is MP8 a functional peroxidase (it catalyzes the oxidation of various substrates by hydrogen peroxide), it has also served as a useful calibration for the interpretation of the electronic spectroscopic properties of heme proteins. NMR structural characterization of MP8 has been difficult, owing to extensive aggregation at millimolar concentrations. We have obtained well-resolved 1H and 13C NMR spectra of monomeric ferric MP8-CN in mixed aqueous−organic solvent mixtures containing excess cyanide. Most peptide resonances were assigned by through-bond correlations using TOCSY spectra; heme resonances were identified largely by through-space correlations using NOESY spectra. HMQC spectra were interpreted with the aid of proton assignments to identify 13C resonances. Most peptide resonances appear within the diamagnetic region and are very sharp, the exceptions being resonances associated with the His18 residue. Protons on the His18 imidazole ring exhibit very broad resonances, reflecting efficient relaxation. The signals of heme substituents are shifted outside of the diamagnetic envelope, with heme methyl resonances appearing between 10 and 25 ppm. The pattern of MP8-CN heme methyl resonances bears a striking resemblance to those of intact cyanoferric heme c protein derivatives. Large amide-proton/α-proton coupling constants and interresidue NOE contacts were found between residues 14 and 18, while moderate amide-α coupling constants were found between residues 19 and 21. The imidazole group of His18 remains coordinated to the ferric center in MP8; and the pattern of heme methyl resonances confirms that a fixed axial imidazole orientation is preserved in the isolated heme active site. The observed interresidue NOE's and amide−H/Hα proton coupling constants indicate that His18 is part of a rigid loop of five residues anchored to the heme that serves to orient the axial imidazole, while residues 19−21 form a flexible C-terminal domain.

Drug-protein interactions: Two-site binding of heterocyclic ligands to a monomeric hemoglobin

The reactivity response of the heme proteins to the heterotropic effectors, purine, caffeine, theophylline, and (C 2H 5) 4N +, have been examined. The heterotropic effectors influence the heme ligation affinities. The heme axial ligation of pyridine and pyrazole have not influenced the hemoglobin's affinity for caffeine and theophylline. The imidazole ligation indicates a mutual interaction between the heme active site and the noncoordinate binding site.

Conserved Glu at the Cytochrome P450 1A2 Distal Site Is Crucial in the Nitric Oxide Complex Stability

Nitric oxide synthase (NOS) has a thiolate-coordinated heme active site similar to that of cytochrome P450 (P450). Both NOS and P450 form stable nitric oxide (NO)-ferric heme complexes, whereas an NO-ferric heme complex of methemoglobin, that has an imidazole-coordinated heme active site, is easily reduced. The NO complex stability of the thiolate-coordinated hemoproteins, however, appeared irreconcilable with the strong electron-donating capability of the cysteine thiolate. In the present study, NO bindings to cytochrome P450 1A2 (P450 1A2) distal mutants were studied in the presence of various substrates. We found that a mutation at Glu-318 to Ala in the putative distal site of P450 1A2, suggested to be important in the O2 activation of P450 reactions, markedly facilitates the reduction of the NO-ferric complex. Addition of 1,2:3,4-dibenzanthracene or phenanthrene almost abolished the mutation effect on the NO complex. Based on these results, together with other spectral and kinetic data, it is suggested that the NO-ferric complex stability of P450, and perhaps of NOS, is largely ascribed to an ionic bridge between NO and the distal carboxyl group.

Probing the Aromatic‐Donor‐Binding Site of Horseradish Peroxidase Using Site‐Directed Mutagenesis and the Suicide Substrate Phenylhydrazine

The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus peroxidase was more resistant to haem modification and phenyl-radical-based inactivation than HRP-C.

Hemoglobin Induced Apolipoprotein B Crosslinking in Low-Density Lipoprotein Peroxidation

Oxidative modification of human low-density lipoprotein (LDL) is thought to play a major role in the development of atherosclerosis. Free hemin, hemoglobin, myoglobin, and horseradish peroxidase (HRP) were reported in different studies as promoters of LDL lipid oxidation. Based on our previous finding that hemin induced oxidative crosslinking of the LDL protein, apolipoprotein B (apo B) (Y. I. Miller and N. Shaklai (1994)Biochem. Mol. Biol. Int.34, 1121–1129), we compared the ability of free hemin and the above hemoproteins to induce peroxidative modification of apo B using SDS–PAGE. The levels of the final products of lipid peroxidation were determined as thiobarbituric acid-reactive substances. Hemoglobin and myoglobin were found to be as active as free hemin and all these were much more active than the classic peroxidase HRP. Moreover, the products of oxidized apo B differed: hemoglobin, myoglobin, and hemin induced mostly covalent aggregates, while HRP caused fragmentation of apo B. Hemoglobin reactivity was expressed at low H2O2concentrations even in the absence of molecular oxygen. Desferal, along with other antioxidants, inhibited the hemoglobin-induced LDL oxidation independently of its iron-chelating property. The high peroxidative reactivity of hemoglobin is explained by its ability (unlike HRP) to transfer the oxidative equivalents from the heme active site, through the globin, to LDL. The apo B radicals thus formed are terminated, yielding intermolecular crosslinked protein. It is suggested that small amounts of the highly reactive hemoglobin in plasma, suffice to trigger LDL protein oxidation (along with its lipid oxidation), thereby inflict the atherosclerosis precondition.

Isolation and characterization of vibrational spectra of individual heme active sites in cytochrome bc1 complexes from Rhodobacter capsulatus.

Resonance Raman spectra of bc1 complexes and isolated c1 subunit from Rhodobacter capsulatus have been obtained using a variety of excitation wavelengths. Spectra obtained via Q-band excitation of bc1 complexes in different redox states were separated to yield the individual vibrational spectra of each of the three heme active sites. Hemes bH and c1 exhibit vibrational spectra typical of b- and c-type hemes, respectively. In contrast, the spectrum of heme bL is anomalous with respect to those of other hemes b. The isolated spectra were also used to assess the effects of inhibitor binding on the local structural environments of the hemes. Neither antimycin nor myxothiazol binding produces dramatic structural perturbations at the hemes. Heme c1 is completely unaffected by the presence of either inhibitor. The vibrational spectra of hemes bH and bL are slightly altered by antimycin and myxothiazol binding, respectively.

Active Site Structure in Cytochrome c Peroxidase and Myoglobin Mutants: Effects of Altered Hydrogen Bonding to the Proximal Histidine

The globins and peroxidases, while performing completely different chemistry, share features of the iron heme active site: a protoporphyrin IX prosthetic group is linked to the protein by the proximal histidine residue. X-ray absorption spectroscopy provides a method to determine the local structure of iron heme active sites in proteins. Our previous studies using X-ray absorption spectroscopy revealed a significant difference in the Fe-N epsilon bond length between the peroxidases and the globins [for a review, see Powers, L. (1994) Molecular Electronics and Molecular Electronic Devices, Vol. 3, p 211 CRC Press Inc., Boca Raton, FL]. Globins typically have an Fe-N epsilon distance close to 2.1 A while the Fe-N epsilon distance in the peroxidases is closer to 1.9 A. We have proposed [Sinclair, R., Powers, L., Bumpus, J., Albo, A., & Brock, B. (1992) Biochemistry 31, 4892] that strong hydrogen bonding to the proximal histidine is responsible for the shorter bond length in the peroxidases. Here we use site-specific mutagenesis to eliminate the strong proximal hydrogen bonding in cytochrome c peroxidase and to introduce strong proximal hydrogen bonding in myoglobin. Consistent with our hypothesis, elimination of the Asp235-His175 hydrogen bond in CcP results in elongation of Fe-N epsilon from approximately 1.9 to approximately 2.1 A. Conversely, introduction of a similar strong proximal hydrogen bond in myoglobin shortens Fe-N epsilon from approximately 2.1 to approximately 1.9 A. These results correlate well with other biochemical data.

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants.

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.