Studies of ferric heme proteins with highly anisotropic/highly axial low spin ( S = 1/2) electron paramagnetic resonance signals with bis-histidine and histidine-methionine axial iron coordination (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1993
The 1H-NMR hyperfine shift pattern of the heme in a variety of low-spin ferricytochromes b 5 has been analyzed in terms of the angular position of the prosthetic group within a structurally and magnetically-conserved protein matrix. A simple model is presented in which the changes in the spread of the predominantly contact shifted methyl and predominantly dipolar shifted meso-H signals of the heme, as well as shift trends for individual signals, provide sensitive indicators of the orientation of the heme relative to the orbital hole (singly-occupied d orbital), which in turn is related to the rhombic magnetic axes. The invariance of the axial His and non-coordinated residue hyperfine shifts show that it is the heme within a relatively rigid protein matrix, rather than the magnetic coordinate system, which is displaced angularly about the heme normal in order to accommodate variations in the polypeptide, orientation of the heme about the a,y-meso axis, and length of heme carboxylate chains. Native heme shows increased counterclockwise rotation about the heme normal in the order rat--, beef--, chicken ferricytochrome bs, which is attributed largely to increased bulk of a variable sequence hydrophobic cluster consisting of residues 23, 25 and 32. The two alternate heme orientations about the a,y-meso axis are shown to also differ by rotation about the heme normal. A semiquantitative estimate of the degree of angular accommodation based on the spread of the meso-H rhombic dipolar shifts indicate rotations of 2-10 ° . Possible functional consequences of such angular accommodation in relation to the role of these proteins in electron transfer are discussed.
Heme methyl 1 H chemical shifts as structural parameters in some low-spin ferriheme proteins
Journal of Biological Inorganic Chemistry, 1999
The different paramagnetic shifts of the four methyl groups in ferriheme proteins have been described as being due to the effect of the axial ligand nodal plane orientation. An equation, heuristically found and theoretically explained, describing the relation between contact and pseudocontact shifts and the position of the axial ligand(s) has been derived for bishistidine ferriheme proteins and for cyanide-histidine ferriheme proteins. The values of the heuristic parameters contained in the equations were found by fitting the shifts of bovine cytochrome b 5 and several bis-histidine cytochromes c 3 and histidine-cyanide systems. The agreement between the observed and the calculated shifts was found to be good. Therefore, by taking advantage of this study, information on the position of the axial ligands, that can be used as a constraint for structure determination, can be obtained from the shifts of the methyl protons.
Journal of the American Chemical Society, 1999
Solution 1 H NMR spectroscopy has been used to characterize the cyanomet myoglobin complexes of a variety of chemically modified hemins in order to elucidate the importance of hemin peripheral electronic, relative to axial His imidazole-induced, rhombic perturbations in raising the orbital degeneracy of the π-bonding d xz ,d yz orbitals. Variation of the hemin 2-and/or 4-position substituents among hydrogen, ethyl, vinyl, acetyl, and formyl groups leads to conserved molecular structure of the heme pocket and orientation of the major magnetic axis for the heme iron, but systematically perturbed heme methyl contact shift patterns. Two strongly rhombically perturbed hemins with single acetyl groups on either pyrrole I or II exhibit heme methyl contact shift patterns and characteristic deviations from Curie law that are very similar to that induced in pseudosymmetric hemins upon incorporation into metMbCN in the alternate orientations about the R,γ-meso axis. The perturbation due to the 4-acetyl group and the axial His bond leads to increased contact shift spread and stronger deviations from Curie behavior compared to WT, indicative of an increased d xz /d yz spacing relative to WT. In contrast, the perturbation due to the 2-acetyl group and axial His nearly cancel, leading to a highly compressed methyl contact shift spread and weaker deviations from Curie behavior than WT. It is shown, moreover, that the larger d xz /d yz splitting with 4-acetylhemin, and the smaller splitting with 2-acetylhemin, relative to WT, result in the expected increase and decrease, respectively, for the axial His contact shift relative to WT. Comparison of the methyl shifts for 16 peripherally modified hemins as model compounds and incorporated into metMbCN shows that the rhombic influences are additive in each of the complexes. Thus, the present results show that chemical functionality of the heme periphery contributes to raising the orbital degeneracy of the heme iron and that such influences can account for orbital ground states that are not necessarily aligned with the axial His orientation. The range of variant 2-and/or 4-substitutions have led to equilibrium heme orientations that are largely the same as found in WT Mb, except for a 4-ethyl group, which favors the reversed heme orientation by 2:1
NMR and DFT Investigation of Heme Ruffling: Functional Implications for Cytochrome c
Journal of the American Chemical Society, 2010
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 π orbitals, which destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the β-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. bren@chem.rochester.edu. ‡ Current address:
Inorganic Chemistry, 2006
Evidence is presented demonstrating that the magnitudes of the 13 C chemical shifts originating from heme meso carbons provide a straightforward diagnostic tool to elucidate the coordination state of high-spin heme proteins and enzymes. Pentacoordinate high-spin heme centers exhibit 13 C meso shifts centered at approximately 250 ppm, whereas their hexacoordinate counterparts exhibit 13 C shifts centered at approximately −80 ppm. The relatively small spectral window (400 to −100 ppm) covering the meso-13 C shifts, the relatively narrow lines of these resonances, and the availability of biosynthetic methods to prepare 13 C-labeled heme (Rivera, M.; Walker, F. A. Anal. Biochem. 1995, 230, 295−302) make this approach practical. The theoretical basis for the distinct chemical shifts observed for meso carbons from hexacoordinate high-spin hemes relative to their pentacoordinate counterparts are now well understood (Cheng, R.-J.; Chen, P. Y.; Lovell, T.; Liu, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 6774−6783), which indicates that the magnitude of the meso-carbon chemical shifts can be used as a simple and reliable diagnostic tool for determining the coordination state of the heme active sites, independent of the nature of the proximal ligand. Proof of the principle for the 13 C NMR spectroscopic approach is demonstrated using hexa-and pentacoordinate myoglobin. Subsequently, 13 C NMR spectroscopy has been used to unambiguously determine that a recently discovered heme protein from Shigella dysenteriae (ShuT) is pentacoordinate.
Biochemistry, 1991
H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b5 reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl)8_,porphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains a t interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b5 is judged much less polar and less sterically accommodating than that of myoglobin. The propionate occupational preference was greatest as the native 7-propionate site, but also possible at the nonnative crystallographic 5-methyl or 8-methyl positions. Only for a propionate a t the crystallographic 8-methyl position was a signifiant perturbation of the native molecular/electronic structure observed, and this was attributed to an alternative propionate-protein hydrogen bond a t the crystallographic 8-methyl position. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links a t either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.
The role of the heme propionates in heme biochemistry
Journal of Inorganic Biochemistry, 2006
There are numerous studies, relying on both experimental and theoretical observations, illustrating the active role of the heme propionates in regulating electron delivery to the iron center as well as biochemical properties of the heme. Evidences for this come from a wide variety of heme containing systems: cytochromes, heme peroxidases, globins, etc. Here, we shortly summarize these studies and revisit previous theoretical calculations (V. Guallar, M.H. Baik, S.J. Lippard, R.A. Friesner, Proc. Natl. Acad. Sci. USA 100 6998-7002) where the propionate groups induced the delocalization of the spin density in the cytochrome P450cam putative active species, Compound I. We introduce novel data, obtained by means of mixed quantum mechanics and molecular mechanics methods, indicating a larger electron delocalization into the protein. We also present novel results based on the recent migration of spin density observed by Barrows et al. (T.P. Barrows, T.L. Poulos, Biochemistry 44 (2005) 14062-68) on an ascorbate peroxidase mutant. All this data strongly supports the importance of the propionate groups in tuning the heme electronic properties.
Febs Letters, 1993
Different monohemic c-type cytochromes were analyzed by visible, EPR and 'H NMR spectroscopies. While the visible and NMR data show unambiguously that the heme iron has a Met-His heme axial coordination, the EPR data indicate an axial hgand field typical of that for a bis-histidinyl ligation. The validity of the widely used EPR methods for the determination of the heme iron axial coordination, based on the crystal field parameters (tetragonality and rhombicity), is questioned.