High-pressure nuclear magnetic resonance studies of hemoproteins. Pressure-induced structural changes in the heme environments of ferric low-spin metmyoglobin complexes

In order to gain an insight into nonbonded interactions in the heme microenvironments of hemoproteins, proton NMR spectra of the cyanide and methylamine complexes of metmyoglobin and its derivatives reconstituted with deutero- and meso-hemins in H2O were studied under high pressures. The exchangeable NH proton of distal histidyl imidazole exhibits substantial pressure-induced shift while the proximal histidyl NH proton shows no pressure effect for the cyanide complexes. The heme peripheral proton signals, especially 5- and 8-methyl and vinyl C alpha H resonances, were also affected by pressure. These observations are interpreted as arising from pressure-induced structural changes in the heme crevice in which the pressure effects are localized to the distal side rather than the proximal side and from possible changes in the van der Waals contacts at the heme periphery with nearby amino acid residues.     

Micro-Raman characterization of high- and low-spin heme moieties within single living erythrocytes

Raman microspectroscopy was applied to monitor porphyrin perturbation associated with the tense-relaxed (T --> R) state transition of hemoglobin (Hb) within a single red blood cell. The spectra exhibited dramatic changes in the spin state region (1650-1500 cm(-1)) and the methine C-H deformation region (1250-1200 cm(-1)) between the 27- and 54-min interval following reoxygenation. Principal components analysis revealed a sigmoidal curve when the PC1 scores versus time were plotted. The inflection point on the curve corresponded to the T --> R transition point spectrum, indicating the cell is composed of Hb in a mixture of spin states. The results of this work illustrate the dynamics of porphyrin perturbation during erythrocyte respiration and may have applications in the diagnosis and treatment of red blood cell disorders including thalassemia and sickle cell anemia.     

The action of cyanogen bromide on horse-heart cytochrome c and horse-heart myoglobin

1. The effects of cyanogen bromide on horse-heart cytochrome c and horse-heart myoglobin have been investigated. Cytochrome c yielded four fragments, of which two were haemopeptides. The two colourless peptides had amino acid compositions corresponding to those that are expected, on the basis of the sequence proposed for horse-heart cytochrome c by Margoliash, Smith, Kreil & Tuppy (1961), from cleavage at both methionine residues. Of the two haemopeptides, one was isolated and shown to be that derived from cleavage at only one methionine residue, that nearer to the C-terminus of the peptide chain. 2. Myoglobin also gave four peptides, three of which accounted for the total amino acid content of the intact protein. The fourth fragment arose by cleavage at a single methionine residue, that nearer the C-terminus. Characterization of this fourth fragment made it possible to deduce the order of arrangement of the fragments in the intact molecule.     

The acid-alkaline transition of a sea turtle myoglobin: coexistence at high pH of high- and low-spin forms

The microenvironment of the iron in a sea turtle Dermochelys coriacea myoglobin is studied using the spectroscopic techniques EPR and optical absorption. Optical absorption spectra in the visible region suggest a great homology between turtle Mb and other myoglobins, such as those from whale, human and elephant. The pK of the acid-alkaline transition is 8.4 slightly lower than the pK of whale and equal to that of elephant myoglobin. The EPR spectrum at pH 7.0 is characteristic of a high-spin configuration with axial symmetry (gx = gy = 5.95). At higher pH, this signal changes in a way different from that observed for whale myoglobin. We observe for turtle Mb both the formation of a low-spin configuration with rhombic symmetry (gx = 2.56, gy = 2.20, gz = 1.90) and of a high-spin species with rhombic distortion (gx = 6.79, gy = 5.18, gz = 2.12). This suggests a lowering of symmetry at the haem, so that now the x and y directions are no more equivalent. This can be explained by amino acid substitution at the distal positions of haem or to off-axial positioning of distal residues. The coexistence at high pH (pH 11.0) of these two spin forms could be explained by the existence of two protein conformations, in which the crystal field splitting factor, delta, and the electron exchange energy are of the same order, allowing the presence of different configurations simultaneously. The presence of different kinds of haem is ruled out by the experiments with nitrosyl turtle Mb and turtle Mb-F showing spectra very similar to those of whale myoglobin. The pk of the acid-alkaline transition, 8.5, obtained from EPR spectra, agrees very well with results from optical absorption.     

Circular dichroism studies of low-spin ferric cytochrome P-450CAM ligand complexes

Circular dichroism (CD) spectroscopy has been used to probe the active site of bacterial ferric cytochrome P-450CAM. The endogenous sixth ligand to the heme iron has been displaced by an extensive series of exogenous oxygen, nitrogen, sulfur and other neutral and anionic donor ligands in an attempt to examine systematically the steric and electronic factors that influence the coupling of the heme chromophore to its protein environment. General trends for each ligand class are reported and discussed. Both the wavelengths and the intensities of the CD bands vary with ligand type and structure. All but one of the complexes exhibit negative CD maxima in their delta and Soret bands. Comparison to ferric myoglobin-thiolate complexes indicates that the negative sign observed for the cytochrome P-450 spectra is not a property of the thiolate fifth ligand, but rather arises from a different interaction of the cytochrome P-450 heme with its protein environment. Complexes with neutral oxygen donors display CD spectra that most closely resemble the spectrum of the native low-spin enzyme. Hyperporphyrin (split Soret) cytochrome P-450 complexes with thiolates, phosphines and cyanide trans to cysteinate have complex CD spectra, reflecting the intrinsic non-degeneracy of the Soret pi pi transitions. The extensive work presented herein provides an empirical foundation for use in analyzing the interaction of heme chromophores with their protein surroundings, not only for the cytochrome P-450 monooxygenases but also for heme proteins in general.     

Spectroscopic properties of low-spin ferrous heme complexes and hemeproteins at 77degrees K.

The low temperature optical spectra in the region of the Q₀₀ (α-band) and Q₀₁ (β-band) transitions of model heme complexes for $\text{b}$- and c-type cytochromes were measured and the results discussed in terms of the similarities and differences to the spectra of horse heart cytochrome $\text{c}$ and other hemeproteins. Comparisons of the resolved vibronic components of the Q₀₁ and β′ bands were made to the recent resonance Raman spectra of hemeproteins. Tentative assignment of the β′ band to Q₀₂ type transitions has been proposed.     

Thermal broadening of the Soret band in heme complexes and in heme-proteins: role of iron dynamics

We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300–20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset of substate interconversions in these latter systems can be reflected in motions of the iron atom with respect to the porphyrin plane. Correspondence to: L. Cordone     

1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl α-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole π bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

Characterization of the different peripheral light-harvesting complexes from high- and low-light grown cells from Rhodopseudomonas palustris

In this paper we demonstrate that the spectroscopically different peripheral light-harvesting complexes from Rhodopseudomonas palustris, strain 2.6.1, isolated from high- and low-light grown cells have widely differing bacteriochlorophyll a (BChl a) resonance Raman spectra in the high-frequency carbonyl region (1550-1750 cm-1). Complexes synthesized in low-light grown cells exhibit Raman spectra characteristic of B800-850 and B800-820 complexes, depending on the excitation conditions. The in vivo strategy for low-light adaptation in this bacterium is thus somewhat different from that generally encountered in the Rhodospirillaceae. In these bacteria, as typified by Rps. acidophila and Rps. cryptolactis, low-light conditions induce the synthesis of B800-820 only complexes in which the hydrogen bonds between the acetyl carbonyl and the B850 binding pocket are broken, inducing changes in the absorption properties of the monomeric bacteriochlorophylls. In the case of Rps. palustris, additional spectral effects occur due to the coupling of the electronic levels of the differently interacting dimers. The extensive use of differential alpha/beta-polypeptide expression [Tadros et al. (1993) Eur. J. Biochem. 217, 867-875] thus allows Rps. palustris to alter its BChl a binding site environments causing the observed spread of BChl a Qy transitions, ranging from 801 to 856 nm.     

Antioxidant properties of oxy- and metmyoglobin]

Lipid peroxide formation is inhibited by met- and oxymyoglobin. Oxymyoglobin as higher antioxidant activity than metmyoglobin. Oxymyoglobin has higher antioxidants activity in concentration of 800 ng/ml than metmyoglobin by 38.6%.     

Synthesis, crystal structures and properties of iron(III) complexes exhibited both high-spin and low-spin states within the crystal lattice

The reaction of FeCl₃ · 6H₂O, potassium hydrotris(pyrazolyl)borate (KTp) and KSCN gives [FeTp₂][TpFe(NCS)₃] (1) and [FeTp₂]₃[Fe(NCS)₆] (2), respectively. The bond lengths and angles indicate that both complexes have [FeTp₂]⁺ cations where Fe(III) ions are in typical low-spin state, and in counter ions, [TpFe(NCS)₃]⁻ for 1 and [Fe(NCS)₆]³⁻ for 2 are both in high-spin state. Variable temperature magnetic susceptibility and ESR results also show that there are double spin states of iron(III) ions within the crystal lattice of both compounds.