Heme Distortion Research Articles

Conserved nonplanar heme distortions in cytochromes c.

A nonplanar distortion of the heme of c-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available high-resolution X-ray crystal structures. This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion. This asymmetry in the heme distortion is also conserved. For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed. Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (> or = 2.00 A) is energetically unfavorable and can occur only in the presence of significant environmental perturbations. Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochrome c, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure. It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties of c-type cytochromes.

Calculation of atom-centered partial charges for heme

Atom-centered partial charges are calculated for the Fe-heme in cytochrome P450cam for use in molecular dynamics simulations of polar substrates bound in the active site of the enzyme. Charges are fit to the electrostatic potential produced by ab initio UHF wavefunctions for an Fe-porphine model. Basis set dependence of these charges is observed using the LANL1DZ, LANL2DZ and augmented 6–31G levels of theory. Upon geometry optimization of the enzyme, these charge sets cause varying degrees of distortion of the porphyrin from its crystallographically observed conformation. Scaling the charges calculated from the augmented 6–31G basis by 75% reduces the heme distortion while preserving reasonable interactions with a polar substrate. A comparison of the calculated charges with other published values is presented.

Raman dispersion spectroscopy probes heme distortions in deoxyHb-trout IV involved in its T-state Bohr effect

The depolarization ratios of heme protein Raman lines arising from vibrations of the heme group exhibit significant dependence on the excitation wavelength. From the analysis of this depolarization ratio dispersion, one obtains information about symmetry-lowering distortions deltaQ(Gamma) of the heme group that can be classified in terms of the symmetry races Gamma = A(1g), B(1g), B(2g), and A(2g) in D(4h) symmetry. The heme-protein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin derivates), which gives rise to specific static heme distortions for each protonation state. From the Raman dispersion data, it is possible to obtain parameters by fitting to a theoretical expression of the Raman tensor, which provide information on these static distortions and also about the pK values of the involved titrable side chains. We have applied this method to the nu(4) (1,355 cm(-1)) and nu(10) (1,620 cm(-1)) lines of deoxygenated hemoglobin of the fourth component of trout and have measured their depolarization ratio dispersion as a function of pH between 6 and 9. From the pH dependence of the thus derived parameters, we obtain pK values identical to those of the Bohr groups, which were earlier derived from the corresponding O(2)-binding isotherms. These are pK(alpha1) = pK(alpha2) = 8.5 for the alpha and pK(beta1) = 7.5, pK(beta2) = 7.4 for the beta chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, the nu(4) mode mainly probes distortions from interactions between the proximal histidine and atoms of the heme core (i.e., the nitrogens and the C(alpha) atoms of the pyrroles). Group theoretical argumentation allows us to relate specific changes of the imidazole geometry as determined by its tilt and azimuthal angle and the iron-out-of-plane displacement to distinct variations of the normal distortions deltaQ(Gamma) derived from the Raman dispersion data. Thus, we found that the pH dependence of the heme distortions deltaQ(A1g) (totally symmetric) and deltaQ(B1g) (asymmetric) is caused by variations of the azimuthal rather than the tilt angle of the Fe-His (F8) bond. In contrast to this, the nu(10) line mainly monitors changes resulting from the interaction between peripheral substituents of the porphyrin macrocycle (vinyl). From the pH dependence of the parameters, it is possible to separately identify distortions deltaQ(Gamma) affecting the hemes in the alpha and beta chains, respectively. From this, we find that in the alpha subunit structural changes induced on protonation of the corresponding Bohr groups are mainly transferred via the Fe-N(epsilon) bond and give rise to changes in the azimuthal angle. In the beta subunit, however, in addition, structural changes of the heme pocket arise, which most probably result from protonation of the imidazole of the COOH-terminal His (HC3 beta). This rearranges the net of H bonds between His HC3 beta, Ser (F9 beta), and Glu (F7 beta).

Investigation of heme distortions and heme-protein coupling in the isolated subunits of oxygenated human hemoglobin by resonance Raman dispersion spectroscopy

To probe the distortions of the heme groups resulting from heme-apoprotein interaction in the isolated subunits of oxygenated human hemoglobin (i.e., alpha SH-oxyHbA and beta SH-oxyHbA), the dispersion of the depolarization ratio of the Raman lines at 1375 cm-1 (nu 4) and 1638 cm-1 (nu 10) was measured at various pHs. The data were analyzed in terms of vibronic coupling parameters which depend on symmetry-classified normal distortions of the heme groups. In the alpha-chain the nu 10 mode is not affected by symmetry-lowering distortions. In the beta-chain, however, this mode is significantly influenced by asymmetric B1g and B2g distortions. This was interpreted in terms of different interactions between the peripheral substituents and the porphyrin macrocycle in the respective chains. The nu 4 mode of both chains is subject to B1g (B2g) and A2g distortions, which are more pronounced in beta SH-oxyHbA. This is most probably due to differences in the repulsive interactions between the proximal imidazole and the pyrrole. While the depolarization ratio of both lines investigated is pH-independent in alpha SH-oxyHbA, it exhibits a significant pH dependence in beta SH-oxyHbA. This parallels the finding that the isolated beta-chains exhibit a Bohr effect whereas the alpha-chains do not. Consequently, the pH dependence of the coupling parameters and the Bohr effect of beta SH-oxyHbA could be rationalized in terms of the very same proton binding processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron paramagnetic resonance and optical spectroscopic study of the heme–human serum albumin complex

Heme–human serum albumin (heme–HSA) complex is paramagnetic because of the presence of a Fe 3+ ion chelated into the protoporphyrin core [1, 2]. At 77 K or lower, the ferric heme in the complex is characterized by a high spin (s = 5 2 electronic configuration. However, the tetragonal symmetry usually displayed by high spin heme iron which is responsible for two EPR lines situated at g⊥ ≅ 6 and g∥ ≅ 2 (fig. 1a) appears to be rhombically distorted upon binding by HSA (Fig. 1b). This distortion shows splitting of the g⊥ ≅ 6 resonance into two resolvable g-values, g x and g y (insert of Fig. 1). The EPR spectrum is interpreted in terms of the spin hamiltonian: H = DS 2 z − 1 3S(S + 1) + E(S 2 x − S 2 y) + β eH̄ ·ĝ· S̄ where E/D = (g x − g y)/48 is, in the present case, equal to 5.35 × 10 −3, corresponding to a rhombicity [3] of 1.61%. Departure from tetragonality may be brought about by several reasons, e.g. by mechanical distortion of heme, perturbation of the heme π-electron distribution, or by π-electron binding to the iron at the 5th and 6th ligand positions [3]. ▪ ▪ ▪ Very little is known about the molecular structure of the complex and, in particular, about the heme or HSA functional groups involved in the binding. However, engagement of the axial positions of heme iron in the binding has been questioned and the hypothesis that the propionyl side chains are involved in this binding has been put forward [4, 5]. Our study is devoted to gain further information on the molecular aspects of the interaction between heme and HSA, in order to understand why HSA binds so tightly the heme groups (causing distortion of the iron symmetry), although it accomplishes a carrier function. The following results were obtained: 1. Absorption optical spectroscopy, in the wavelength range between 300 and 1100 nm, of heme alone (Fig. 2b) and of heme–HSA complex (Fig. 2a) in DMSO/H 2O solvent (in this solvent it has been ▪shown that hemes are monomeric and HSA is not denaturated [4]) permitted us to ascertain definitively that at least one axial position of iron is involved in the binding; 2. Spin labeling of the lone sulfhydryl group at position 34 of the protein, by a maleimide nitroxide (Fig. 3), led us to estimate using Leigh's theory a a distance of about 10 Å between the paramagnetic ferric heme ion and the bound spin label; 3. The use of Fe 3+-protoporphyrin, spin labeled at its propionyl carboxyl groups, provided strong evidence that these groups are directly involved in the formation of the complex; probably via hydrogen bonds. 4. Emission optical spectroscopy showed that progressive quenching of the lone tryptophan (at position 214) fluorescence occurred during the formation of the complex (Fig. 4). According to the expression 6: ▪ the distance r between the heme group and tryptophan was estimated to be very close to 1.5 nm. Taking into account these results, and the fact that KCN converted the complex to a low spin form only at high concentrations, we can imagine that the heme group is bound in a rather hydrophobic pocket to HSA, which provides a ligand to the 5th axial position of iron and further hydrogen bonds to the propionyl side chains. The pocket is situated very close to the SH group and the close-lying aromatic tryptophan is probably responsible for the rhombic distortion, via a perturbation of the heme π-electron distribution [7]. Further thermodynamic results suggest the occurrence of an entropy-controlled internalization process of the bound heme, to provide steric protection against diffusion off the carrier protein surface, thus allowing for efficient release only at the target.

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.