Heme Methyl Resonances Research Articles

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (-96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (-89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.

Gd(III) Chelates as NMR Probes of Protein–Protein Interactions. Case Study: Rubredoxin and Cytochromec3

Two cyclen-derived Gd probes, [Gd-DOTAM](3+) and [Gd-DOTP](5-) (DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide; DOTP = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), were assessed as paramagnetic relaxation enhancement (PRE)-inducing probes for characterization of protein-protein interactions. Two proteins, Desulfovibrio gigas rubredoxin and Desulfovibrio gigas cytochrome c(3), were used as model partners. In a (1)H NMR titration it was shown that [Gd-DOTP](5-) binds to cytochrome c(3) near heme IV, causing pronounced PREs, characterized by line width broadenings of the heme methyl resonances at ratios as low as 0.08. A K(d) of 23 ± 1 μM was calculated based on chemical shift perturbation of selected heme methyl resonances belonging to three different heme groups, caused by allosteric effects upon [Gd-DOTP](5-) binding to cytochrome c(3) at a molar ratio of 2. The other probe, [Gd-DOTAM](3+), caused PREs on a well-defined patch near the metal center of rubredoxin (especially the patch constituted by residues D19-G23 and W37-S45, which broaden beyond detection). This effect was partially reversed for some resonances (C6-Y11, in particular) when cytochrome c(3) was added to this system. Both probes were successful in causing reversible PREs at the partner binding site, thus showing to be good probes to identify partners' binding sites and since the interaction is reversible to structurally characterize protein complexes by better defining the complex interface.

NMR and DFT Investigation of Heme Ruffling: Functional Implications for Cytochrome c

Out-of-plane (OOP) deformations of the heme cofactor are found in numerous heme-containing proteins and the type of deformation tends to be conserved within functionally related classes of heme proteins. We demonstrate correlations between the heme ruffling OOP deformation and the (13)C and (1)H nuclear magnetic resonance (NMR) hyperfine shifts of heme aided by density functional theory (DFT) calculations. The degree of ruffling in the heme cofactor of Hydrogenobacter thermophilus cytochrome c(552) has been modified by a single amino acid mutation in the second coordination sphere of the cofactor. The (13)C and (1)H resonances of the cofactor have been assigned using one- and two-dimensional NMR spectroscopy aided by selective (13)C-enrichment of the heme. DFT has been used to predict the NMR hyperfine shifts and electron paramagnetic resonance (EPR) g-tensor at several points along the ruffling deformation coordinate. The DFT-predicted NMR and EPR parameters agree with the experimental observations, confirming that an accurate theoretical model of the electronic structure and its response to ruffling has been established. As the degree of ruffling increases, the heme methyl (1)H resonances move upfield while the heme methyl and meso (13)C resonances move downfield. These changes are a consequence of altered overlap of the Fe 3d and porphyrin pi orbitals, which destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the beta-pyrrole and meso carbons, respectively. Consequently, the heme ruffling deformation decreases the electronic coupling of the cofactor with external redox partners and lowers the reduction potential of heme.

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH

The ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN−) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0–7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemical exchange cross peaks appear in the nuclear Overhauser effect spectroscopy/exchange spectroscopy (NOESY/EXSY) spectra for the heme methyl resonances at low pH (pH* 4.0–5.5), which indicate chemical exchange between two species. We have shown these to be two different exogenous ImH or Hm orientations that are denoted B and B′, with the ImH plane nearly parallel and perpendicular to the ImH plane of the protein-provided His57, respectively. The wt NP2–CN complex also shows EXSY cross peaks due to chemical exchange, which is shown to be a result of interchange between two ruffling distortions of the heme. The same ruffling distortion interchange is also responsible for the ImH and Hm chemical exchange. For the three NP2(V24E) ligand complexes, no EXSY cross peaks are observed, but the A:B ratios change dramatically with pH. The fact that heme favors the A orientation highly for NP2(V24E) at low pH as compared with wt NP2 is believed to be due to the steric effect of the V24E mutation. The existence of the B′ species at lower pH for wt NP2 complexes and the increase in A heme orientation at lower pH for NP2(V24E) are believed to be a result of a change in structure near Glu53 when it is protonated at low pH. 1H{13C} heteronuclear multiple quantum coherence (HMQC) spectra are very helpful for the assignment of heme and nearby protein side chain resonances.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-009-0551-3) contains supplementary material, which is available to authorized users.

NMR Studies and Redox Titration of the Tetraheme Cytochrome c3 from Desulfomicrobium baculatum

The tetraheme cytochromes c3 isolated from two strains of Desulfomicrobium baculatum were studied by monitoring the spectral changes undergone during redox titrations followed by 1H NMR. The evolution of the three-proton intensity signals at low field allowed the partial identification of the heme methyl resonances in the spectrum of the fully oxidized state. The chemical shift variation shown by the protons of the aromatic sidechains as well as of the substituents of the higher-potential heme HIII [Coutinho, I. B., Turner, D. L., LeGall, J. & Xavier, A. V. (1993) Biochem. J. 294, 899–908] yielded the assignment of the lower midpoint redox potential to heme HII in the three-dimensional structure. This cross-assignment is achieved by comparing the chemical shifts of the resonances in the spectra obtained at intermediate oxidation levels with the pseudocontact shifts predicted to arise from the three lower-potential hemes. The cross-assignment for the cytochromes from these two strains is different from that of the cytochromes from Desulfovibrio vulgaris and Desulfovibrio gigas.

A Copper Protein and a Cytochrome Bind at the Same Site on Bacterial Cytochrome c Peroxidase

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome.

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.

Insights into the alkaline transformation of ferricytochrome c from (1)H NMR studies in 30% acetonitrile-water.

Recently, we found that ferricytochrome c (ferricyt c) undergoes significant structural changes in mixed aqueous-nonaqueous media, resulting in the formation of a mixture of alkaline-like species. The equilibrium composition of this mixture of species is dependent on the dielectric constant of the mixed solvent medium. One-dimensional (1D) and two-dimensional (2D) (1)H nuclear magnetic resonance (NMR) methods have now been used to study these alkaline-like forms in 30% acetonitrile-water solution. A native-like (M80-ligated) III* form, two lysine-ligated forms (IVa* and IVb*), and a hydroxide-ligated form (V*) were observed. Heme proton resonance assignments for these forms were accomplished using 1D (1)H NMR and 2D nuclear Overhauser effect spectroscopy methods at 20 degrees C and 35 degrees C. The chemical exchange between the alkaline forms in 30% acetonitrile solution facilitated heme proton resonance assignments. Based on examination of the heme proton chemical shifts and several highly conserved amino acid residues, the electronic structure, secondary structure, and hydrogen bond network in the vicinity of the heme in the III* form were found to be intact. Similarly, the heme electronic structure of the IVa* form was found to be comparable to that of the IVa form. Differences in the order of the heme methyl resonances in the IVb* form, however, suggest that the heme active site in this form is somewhat different from that observed in aqueous alkaline solution. In addition, resonance assignments for the 8- and 3-methyl heme protons were made for the hydroxide-ligated V* form for the first time. The observation of chemical exchange peaks between all species except IVb* and IVa* or V* was used to propose an exchange pathway between the different forms of ferricyt c in 30% acetonitrile solution. This pathway may be biologically significant because ferricyt c, which resides in the intermembrane space of mitochondria, is exposed to medium of relatively low dielectric constant when it interacts with the mitochondrial membrane.

1H NMR study of 2-methylimidazole binding to cytochrome c: a comprehensive investigation of the role of the methyl substituent on the ligand binding affinity and heme electronic structure in imidazole–cytochrome c complexes

The binding of 2-methylimidazole (2mim) to horse heart cytochrome c (hh cyt c) has been studied by NMR spectroscopy. Some proton resonances were assigned and the kinetic and thermodynamic parameters for the binding were presented. Based on its unique hyperfine shift pattern and the anomalous temperature dependence of the heme methyl resonances, the heme electronic structure was discussed and the orientation of the bound 2mim was estimated. With these data, a comprehensive comparison of imidazole (Him), 4-methylimidazole (4mim) and 2mim bound cyt c complexes was made on the ligand binding affinity and the heme electronic structure. Different properties in these ligand–cyt c complexes provide a useful lesson for the study of protein–small molecular interactions.

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.

Anion Binding to Cytochromec2: Implications on Protein–Ion Interactions in Class I Cytochromesc

The binding of several inorganic and carboxylate anions to cytochromec2fromRhodopseudomonas palustrishas been investigated by monitoring the salt-induced changes in the redox potential of the heme, using an interpretative model based on the extended Debye–Hückel equation. Most anions were found to interact specifically with the protein at one or multiple sites. Binding constants to the oxidized protein in the range 101–102m−1were determined from the anion concentration dependence of the chemical shift of the isotropically shifted heme methyl resonances. For several anions the stoichiometry and strength of the binding to cytochromec2were found comparable with those determined for mitochondrial cytochromesc, in spite of the limited sequence similarity (less than 40%) and the lower positive charge of the bacterial protein. These analogies were interpreted as indicative of the existence of common binding sites which are proposed to be located in the conserved lysine-rich domain around the solvent-exposed heme edge, which is also the surface area likely involved in the interaction with redox partners. The changes inE° due to partial neutralization of the positive charge of cytochromec2due to specific anion binding were found comparable with those for the mitochondrial species.

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.

Regulation of the redox order of four hemes by pH in cytochrome C3 from D. vulgaris Miyazaki F

The assignment of 1H-NMR signals of the heme methyl and propionate groups of cytochrome c 3 of D. vulgaris Miyazaki F was performed. The heme assignment was revised for hemes 2 and 3 (sequential heme numbering). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 following it in this order. The p 2H titration of heme methyl signals in four macroscopic oxidation states was performed in the p 2H range of 5.2 to 9.0. While the heme methyl resonances in the fully oxidized state showed just small changes with p 2H most resonances in the intermediate oxidation states revealed clear p 2H dependence. In particular, the methyl resonances of heme 1 shifted significantly in the acidic region. Then, the chemical shifts of β-CH 2 (next to the carboxyl group) of all propionate groups in the fully oxidized state were observed at various p 2H in the range of 4.5 to 9.0. Only the propionate group at C-13 (IUPAC-IUB nomenclature) of heme 1 showed a clear change in this p 2H range, its titration curve being similar to those of the methyl resonances of heme 1 in the intermediate oxidation states. pK a of the propionate group was 5.95 ± 0.05. Analysis of the microscopic formal redox potentials was carried out for the observations at p 2H 5.2, 7.1 and 9.0. The redox potentials of heme 1 showed the most remarkable p 2H dependence, resulting in the change of the order of the redox potentials of four berries. A significant change was also found in the interacting potential between hemes 1 and 2. In the light of the p 2H-titration experiments, the propionate at C-13 of heme 1 was identified as the most plausible ionizable group responsible for the p 2H dependence of microscopic redox potentials of heme 1 in the acidic region.

NMR studies and redox titration of the tetraheme cytochrome c3 from Desulfomicrobium baculatum. Identification of the low-potential heme.

The tetraheme cytochromes c3 isolated from two strains of Desulfomicrobium baculatum were studied by monitoring the spectral changes undergone during redox titrations followed by 1H NMR. The evolution of the three-protein intensity signals at low field allowed the partial identification of the heme methyl resonances in the spectrum of the fully oxidized state. The chemical shift variation shown by the protons of the aromatic sidechains as well as of the substituents of the higher-potential heme HIII [Coutinho, I. B., Turner, D. L., LeGall, J. & Xavier, A. V. (1993) Biochem. J. 294, 899-908] yielded the assignment of the lower midpoint redox potential to heme HII in the three-dimensional structure. This cross-assignment is achieved by comparing the chemical shifts of the resonances in the spectra obtained at intermediate oxidation levels with the pseudocontact shifts predicted to arise from the three lower-potential hemes. The cross-assignment for the cytochromes from these two strains is different from that of the cytochromes from Desulfovibrio vulgaris and Desulfovibrio gigas.

1H NMR characterization of myoglobins where exogenous ligands replace the proximal histidine.

The role of the proximal ligand in determining the structure and ligand binding properties of sperm whale myoglobin has been investigated using the mutant H93G(L), where the proximal histidine has been replaced with glycine, creating a cavity which can be occupied by a variety of exogenous ligands, L, to the iron [Barrick, D. (1994) Biochemistry 33, 6546-6554; DePillis, G.D., Decatur, S.M., Barrick, D., & Boxer, S.G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. In this report, we present the assignments of selected protons of the heme and heme pocket residues in the metcyano complexes of H93G with Im and a series of methyl-substituted Ims [H93G(Im)CN, H93G(N-MeIm)CN, H93G(2-MeIm)CN, H93G-(4-MeIm)CN]. Each complex has a unique 1H NMR spectrum, providing a fingerprint for documenting the ligand exchange phenomenon. Moreover, the identification of NOEs between the protons of proximal ligands and protons of proximal pocket amino acid residues confirms that the new ligand occupies the proximal cavity in solution. The pattern of hyperfine-shifted heme methyl resonances in H93G(Im)CN is very different from that of wild-type Mb, consistent with the differences compared to wild-type in is very different from that of wild-type Mb, consistent with the differences compared to wild-type in orientation of the proximal imidazole observed in the X-ray crystal structure of H93G(Im) [Barrick, D. (1994) Biochemistry 33, 6546-6554]. Addition of deuterated Im to H93G(Im)CN permits direct observation of exchange of proximal ligands with ligands from solution; exchange of Im for deuterated Im in the metcyano complex occurs with half-life of around 10 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a ternary complex formed between flavodoxin and cytochrome c3: 1H-NMR and molecular modeling studies.

Small electron-transfer proteins such as flavodoxin (16 kDa) and the tetraheme cytochrome c3 (13 kDa) have been used to mimic, in vitro, part of the complex electron-transfer chain operating between substrate electron donors and respiratory electron acceptors, in sulfate-reducing bacteria (Desulfovibrio species). The nature and properties of the complex formed between these proteins are revealed by 1H-NMR and molecular modeling approaches. Our previous study with the Desulfovibrio vulgaris proteins [Moura, I., Moura, J.J. G., Santos, M.H., & Xavier, A. V. (1980) Cienc. Biol. (Portugal) 5, 195-197; Stewart, D.E. LeGall, J., Moura, I., Moura, J. J. G., Peck, H.D. Jr., Xavier, A. V., Weiner, P. K., & Wampler, J.E. (1988) Biochemistry 27, 2444-2450] indicated that the complex between cytochrome c3 and flavodoxin could be monitored by changes in the NMR signals of the heme methyl groups of the cytochrome and that the electrostatic surface charge (Coulomb's law) on the two proteins favored interaction between one unique heme of the cytochrome with flavodoxin. If the interaction is indeed driven by the electrostatic complementarity between the acidic flavodoxin and a unique positive region of the cytochrome c3, other homologous proteins from these two families of proteins might be expected to interact similarly. In this study, three homologous Desulfovibrio cytochromes c3 were used, which show a remarkable variation in their individual isoelectric points (ranging from 5.5 to 9.5). On the basis of data obtained from protein-protein titrations followed at specific proton NMR signals (i.e., heme methyl resonances), a binding model for this complex has been developed with evaluation of stoichiometry and binding constants. This binding model involves one site on the cytochromes c3 and two sites on the flavodoxin, with formation of a ternary complex at saturation. In order to understand the potential chemical form of the binding model, a structural model for the hypothetical ternary complex, formed between one molecule of Desulfovibrio salexigens flavodoxin and two molecules of cytochrome c3, is proposed. These molecular models of the complexes were constructed on the basis of complementarity of Coulombic electrostatic surface potentials, using the available X-ray structures of the isolated proteins and, when required, model structures (D. salexigens flavodoxin and Desulfovibrio desulfuricans ATCC 27774 cytochrome c3) predicted by homology modeling.

Binding of horseradish, lignin, and manganese peroxidases to their respective substrates

The present study utilizes 1H NMR spectroscopy to characterize the binding of substrate to heme active site of three different peroxidases, horseradish peroxidase, lignin peroxidase, and manganese peroxidase. Information has been obtained on the site of p-cresol binding to the active-site cavity of the cyanide derivative of horseradish peroxidase. This information was obtained by relaxation enhancements of the substrate protons and connectivities between the latter and heme 8-CH3 and a Phe residue. Manganese(II) is shown to bind to ferri-manganese peroxidase and its cyanide derivative in a specific site with a high-affinity constant (10(4) M-1). Manganese(II) binding exhibits a slow exchange rate with respect to the difference in T2(-)1 of the affected signals in the manganese(II)-containing and manganese(II)-free species. Manganese(II) affects the line width of certain heme methyl resonances and of certain one-proton intensity signals in manganese peroxidase and its cyanide derivative. The behavior of MnP toward manganese(II) is compared to that of the closely related peroxidase, lignin peroxidase (LiP), with its native substrate veratryl alcohol. LiP does not have a specific binding site for manganese(II).

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis

The reduction of the tetraheme cytochrome c 3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenborough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theory. Thus, the very low reduction potentials of the cytochrome c 3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c 3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149–151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.

Heme methyl hyperfine shift pattern as a probe for determining the orientation of the functionally relevant proximal histidyl imidazole with respect to the heme in hemoproteins

Heme methyl 1H and 13C resonances of met-cyano form of myoglobin from the shark, Geleorhinus japonicus (GJMbCN), have been assigned via 1H- 13C heteronuclear shift correlated spectroscopy (COSY) connectivities and their hyperfine shifts were compared with those of the corresponding resonances of some hemoproteins. Variation of the heme methyl 1H hyperfine shift pattern correlates well with the angle (Φ) between the projection of the proximal histidyl imidazole plane onto the heme plane and the N II-Fe-N iv vector. The alteration of the interaction of the heme peripheral side-chains and/or the iron-bound ligand with the surrounding amino acid residues cannot account for large differences in the shifts of the corresponding heme methyl resonances between GJMbCN and sperm whale MbCN. Since the heme methyl 1H shifts for GJMbCN fall in between those of the corresponding resonances for sperm whale Mb and Aplysia limacma Mb in which the Φ values have been reported to be 19° and 29°, respectively, the Φ value in GJMb is estimated to be slightly larger than 19°.

Proton hyperfine resonance assignments in trimethylphosphine ligated ferrimyoglobin using saturation transfer spectroscopy

1H-NMR saturation transfer spectroscopy and nuclear Overhauser effect (NOE) have been utilized to assign several heme resonances in the low-spin trimethyl phosphine complex of sperm whale metmyoglobin. The two methods permit the location of the heme methyl resonances and the heme 2-vinyl group resonances. A qualitative comparison involving the methyl shift pattern in metMbN 3, metMbCN, imidazole metMb and trimethyl phosphine metMb shows a reverse methyl shift between pyrrole I and pyrrole IV. The different hyperfine shift pattern for metMbPMe 3 is suggested to arise from: (i) a possible reorientation of the proximal histidine plane; (ii) different heme protein contacts in the different ligated proteins, and (iii) a small contribution from high-spin character. The 2-vinyl group is formed in the cis plane orientation.