Dynamics of wild-type HiPIPs: a Cys77Ser mutant and a partially unfolded HiPIP

The temperature dependence of the mean square displacement of the (57)Fe nuclei due to motion faster than 100 ns are measured by temperature-dependent M?ssbauer spectroscopy for oxidized and reduced HiPIPs from Ectothiorhodospira halophila, Chromatium vinosum WT and a Cys77Ser mutant. The behaviour is interpretable in the frame of the general model of protein dynamics distinguishing two temperature intervals. The character of harmonic and quasi-diffusional modes in HiPIPs is discussed. Dynamic information obtained from M?ssbauer spectroscopy and Fe K-edge EXAFS are compared. Structure dynamics of the iron-sulfur cluster in the partially unfolded reduced HiPIP from C. vinosum was investigated by M?ssbauer spectroscopy and EXAFS, indicating an intact metal centre and a protein backbone with a largely collapsed secondary structure. The role of the cofactor during protein folding is discussed. Differences in the dynamics between the native protein and the molten globule are found at physiological temperatures only. The structure and dynamic behaviour of the [Fe(4)S(4)]Cys(3)Ser cluster in the Cys77Ser mutant of the HiPIP from C. vinosum are analysed. The temperature dependence of electron relaxation in oxidized HiPIPs is investigated by M?ssbauer spectroscopy and analysed theoretically, considering spin-spin and spin-lattice relaxation. The latter consists of contributions from direct phonon bottleneck and Orbach mechanisms. The data agree with former pulsed EPR results. Orbach relaxation is interpreted as due to transitions between electronic isomers of oxidized HiPIPs. With this interpretation, the energetic difference between both isomers equals the energy gap estimated from the temperature dependence of the Orbach relaxation.     

Study of protein dynamics using M?ssbauer spectroscopy

Last experimental results of the study of protein dynamics by M?ssbauer absorption spectroscopy and Rayleigh scattering of M?ssbauer radiation are reviewed. Dynamical properties of proteins following from the theoretical treatment of these data are described.     

M?ssbauer spectroscopy, electron microscopy and electron diffraction studies of the iron cores in various human and animal haemosiderins

M?ssbauer spectroscopy has indicated significant differences in the iron-containing cores of various haemosiderins. In the present study, haemosiderin was isolated from a number of animal species including man. In addition, haemosiderin was isolated from patients with primary idiopathic haemochromatosis or with secondary (transfusional) iron-overload. The iron cores of the animal and normal human haemosiderin appear to be very similar by M?ssbauer spectroscopy, and the electron diffraction data indicate a ferrihydrite structure similar to that of ferritin cores. The haemosiderin isolated from secondary iron-overload shows anomalous behaviour in its temperature-dependent M?ssbauer spectra. This can be understood in terms of the microcrystalline goethite structure of the cores as indicated by electron diffraction. The haemosiderin cores obtained in the case of primary haemochromatosis have an amorphous Fe(III) oxide structure and show M?ssbauer spectra characteristic of a magnetically disordered material, which only orders at very low temperatures.     

Study of the relaxation of nonequilibrium hemoglobin states by M?ssbauer spectroscopy

Nonequilibrium hemoglobin states formed at low-temperature (T = 77K) reduction of its derivatives (MetHb and HbO2) by thermolysed electrons have been studied by M?ssbauer spectroscopy. Relaxation of nonequilibrium states at the samples heating was observed. Correlation between the relaxation temperatures and the changes of protein dynamic structure determined from M?ssbauer data were stated.     

M?ssbauer spectroscopic studies of deproteinised, sub-fractionated and reconstituted ferritins: the relationship between haemosiderin and ferritin

In a previous study of human haemosiderin and ferritin by a combination of M?ssbauer spectroscopy and electron microscopy, it was observed that the M?ssbauer spectra of haemosiderin showed a very different temperature dependence to those of ferritin. These differences were related to the superparamagnetic behaviour of small particles of a magnetic material and suggested that the magnetic anisotropy constant of the haemosiderin was considerably larger than that of the ferritin. In the present work, samples of ferritin have been examined by M?ssbauer spectroscopy following partial deproteinisation, subfractionation, and reconstitution with and without phosphate, in order to investigate whether these procedures lead to changes in the magnetic anisotropy constant of the iron-containing cores. There is no evidence from the present data that changes in the protein shell, in the size of the iron-containing cores of ferritin, or in the phosphate content lead to any significant changes in the magnetic anisotropy constant, as obtained from the temperature dependence of the M?ssbauer spectra. These results indicate that the different magnetic anisotropy constant observed in the case of human haemosiderin resulting from transfusional iron overload must arise from other significant differences in the composition or structure of the iron-containing cores.     

M?ssbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Spectral deconvolution of the heme components.

M?ssbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic M?ssbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic M?ssbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic M?ssbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.     

Study of normal human fetal and adult hemoglobins by M?ssbauer spectroscopy

Human normal fetal and adult hemoglobins were investigated by M?ssbauer spectroscopy. The differences discovered in quadrupole splitting are connected with distinctions in protein molecular structures. An approximation of M?ssbauer spectra of hemoglobins in supposition of nonequivalence of Fe(II) electronic structure in alpha-, beta- and gamma-subunits in tetramers was proposed. Qualitative analysis of the relationship of Fe(II) electronic structure, molecular structure of the active site and functionality of hemoglobin was carried out.     

Comparison and characterization of the [Fe4S4]2+/3+ centre in the wild-type and C77S mutated HiPIPs from Chromatium vinosum monitored by Mössbauer, 57Fe ENDOR and EPR spectroscopies

M?ssbauer, 57Fe ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T = 5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by 57Fe ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T = 5 K in native and mutated oxidized HiPIPs to achieve agreement between M?ssbauer and 57Fe ENDOR spectroscopies. The M?ssbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the M?ssbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The M?ssbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. M?ssbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.     

Evidence for a three-iron center in a ferredoxin from Desulfovibrio gigas. M?ssbauer and EPR studies

The tetrameric form of a Desulfovibrio gigas ferredoxin, named Fd II, mediates electron transfer between cytochrome c3 and sulfite reductase. We have studied two stable oxidation states of this protein with M?ssbauer spectroscopy and electron paramagnetic resonance. We found 3 iron atoms/monomer and a spin concentration of 0.9 spins/monomer for the oxidized protein. Taken together, the EPR and M?ssbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The M?ssbauer data show also that this metal center is structurally similar, if not identical, with the low potential center of a ferredoxin from Azotobacter vinelandii, a novel cluster described recently (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796).     

Evidence for polynuclear aggregates of ferric daunomycin. A M?ssbauer, EPR, X-ray absorption spectroscopy and magnetic susceptibility study.

The interaction of the antitumor agent daunomycin (DN) with ferric iron has been analysed by M?ssbauer spectroscopy, EPR, extended X-ray absorption fine structure (EXAFS), and magnetic susceptibility measurements. In contrast to literature data, at millimolar iron and anthracycline concentrations no solitary Fe(DN)3 complexes are formed in appreciable amounts. The M?ssbauer spectroscopic analysis revealed severe dependencies on temperature, on the preparation procedure, the time allowed for equilibration, and on the metal/ligand ratio. The M?ssbauer spectra exhibit two components: a broad magnetic sextet and a quadrupole doublet at an Fe/DN molar ratio of 1:3 and exclusively a doublet at a molar ratio of 1:20, indicating an equilibrium of these two spectral components. The EPR spectra are dominated by a signal at g(eff) = 2. Double integration of the EPR signals enabled the determination of their spin density and a correlation between EPR and M?ssbauer spectra. The M?ssbauer sextet species is EPR invisible and corresponds to magnetically ordered polynuclear aggregates with high magnetic anisotropy. EXAFS and susceptibility measurements provide additional evidence for the formation of polynuclear aggregates of ferric daunomycin. The quadrupole doublet species in the M?ssbauer spectra correlates with the g = 2 signal in EPR. This species is also related to a magnetically ordered system, exhibiting, however, superparamagnetic behavior due to less magnetic anisotropy. Since daunomycin forms dimers in aqueous solution at millimolar concentrations, we conclude that the cooperative phenomena observed in EPR and M?ssbauer spectra are a consequence of its stacking effects.     

Dynamic structural disorder of the FeO<Subscript>2</Subscript> moiety in oxymyoglobin studied by nuclear resonant forward scattering of synchrotron radiation

Oxy- as well as deoxymyoglobin exhibit a pronounced temperature dependence of the quadrupole splitting of the heme iron as detected by conventional M?ssbauer spectroscopy. With nuclear resonant forward scattering (NFS) of synchrotron radiation, which can be viewed as M?ssbauer spectroscopy in the time domain, it is shown that this spectroscopic behavior, although it is phenomenologically similar in the two cases, is based on completely different physical mechanisms. It is demonstrated that stochastic fluctuations of the iron electric field gradient in MbO(2), which are due to the dynamic structural disorder of the FeO(2) moiety, are the reason for the temperature-dependent alterations of the coherent quantum beat pattern in the NFS spectra of MbO(2), in contrast to deoxyMb where transitions between orbital states of iron take place. This subtle spectroscopic difference cannot be inferred from conventional M?ssbauer spectroscopy.     

Spectroscopic studies of the nature of the iron clusters in the soluble hydrogenase from Desulfovibrio desulfuricans (strain Norway 4)

57Fe-enriched samples of the soluble hydrogenase from Desulfovibrio desulfuricans (Norway) have been investigated in both the native (oxidized) and the dithionite-reduced states using M?ssbauer spectroscopy. The data clearly show that the iron in this enzyme is predominantly in the form of iron-sulphur clusters which are closely similar to the [4Fe-4S] clusters found in a large number of ferredoxins, such as that from Bacillus stearothermophilus. There appear to be two [4Fe-4S] clusters. The iron-sulphur clusters in the oxidized protein are virtually diamagnetic, as indicated by M?ssbauer, electron spin resonance and magnetic circular dichroic spectroscopy. On reduction by dithionite + methyl viologen, M?ssbauer spectroscopy showed that only 50% of the [4Fe-4S] clusters were reduced. Even reduction with hydrogen up to a pressure of 23 GPa did not reduce the iron-sulphur clusters completely. An ESR signal due to a rapidly relaxing species with g = 2.03, 1.89 was observed in the reduced protein, together with a weaker spectrum from a slower-relaxing species at g = 2.34, 2.12.     

Mössbauer spectroscopy and ELISA studies reveal differences between Parkinson's disease and control substantia nigra

The possible role of iron in the degeneration of nervous cells in Parkinson's disease (PD) was studied with the use of M?ssbauer spectroscopy (MS) and enzyme-linked immunoabsorbent assay (ELISA). M?ssbauer data were obtained at 90 and 4.1 K from 21 samples of control and 9 samples of parkinsonian substantia nigra (SN). M?ssbauer spectra were very similar to those observed in ferritin. Small differences were detected between the spectra obtained from PD and from control SN, and could be due to a slight difference in the composition of the ferritin-like iron cores or due to the presence of about 8% of non-ferritin-like iron in parkinsonian SN. ELISA studies from 11 controls and 6 parkinsonian SN showed a decrease in the concentration of L-chains in wet tissues of PD-SN compared to control SN. The decrease in the amount of L subunits may correspond to a decreased ability of this ferritin to keep iron in a safe form. Iron released from ferritin or neuromelanin (NM) may be the source of such iron, which may cause the difference in the M?ssbauer spectra and may trigger oxidative stress leading to cell death.     

M?ssbauer investigations of high-spin ferrous heme proteins. II. Chloroperoxidase, horseradish peroxidase, and hemoglobin.

Reduced samples of chloroperoxidase, horseradish peroxidase, and deoxyhemoglobin were studied by M?ssbauer spectroscopy in strong magnetic fields. The intricate paramagnetic spectra of chloroperoxidase were evaluated in detail in the framework of a spin Hamiltonian pertinent to high-spin ferrous iron. The studies strongly suggest that, in their reduced states, chloroperoxidase from Caldariomyces fumago and cytochrome P-450 from Pseudomonas putida have similar, if not identical ligand structures of the heme iron. The spectral similarities of these two proteins, noted in an earlier M?ssbauer investigation, are further explored and substantiated. Reduced horseradish peroxidase and deoxyhemoglobin, on the other hand, show high-field M?ssbauer spectra that differ considerably from each other and, in particular, from those of the P-450 type, suggesting a different ligand arrangement of the heme iron for each case.     

M?ssbauer study of cytochrome c2 from Rhodospirillum rubrum. Sign of the product gxgygz of some low spin ferric heme proteins.

We have studied cytochrome c2 from Rhodospirllum rubrum with M?ssbauer spectroscopy and electron paramagnetic resonance. The M?ssbauer data on the ferric protein, taken in external magnetic fields up to 50 kG, were analyzed within the framework of the ligand field model commonly used to evaluate low-spin ferric heme compounds. The data analysis shows that the determinant of the electronic g-tensor, i.e. the product gxgygz, is positive for cytochrome c2. We have reanalyzed published M?ssbauer data of some low-spin ferric heme proteins with respect to the sign of the g-tensor determinant. We find that gxgygz is also positive for the cytochromes c, bs, and P-450, and for chloroperoxidase.     

M?ssbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa.

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and M?ssbauer spectroscopy. Low temperature M?ssbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has M?ssbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The M?ssbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.     

Evidence for variable metal-radical spin coupling in oxoferrylporphyrin cation radical complexes

Oxoferrylporphyrin cation radical complexes were generated by m-chloroperoxybenzoic acid oxidation of the chloro and trifluoromethanesulfonato complexes of tetramesitylporphyrinatoiron(III) [(TMP)Fe] and the trifluoromethanesulfonato complex of tetra(2,6-dichlorophenyl)porphyrinatoiron(III) [TPP(2,6-Cl)Fe]. Coupling between ferryl iron (S = 1) and porphyrin radical (S' = 1/2) spin systems was investigated by M?ssbauer and EPR spectroscopy. The oxoferrylporphyrin cation radical systems generated from the TMP complexes show strong ferromagnetic coupling. Analysis of the magnetic M?ssbauer spectra, using a spin Hamiltonian explicitly including a coupling tensor J, suggests an exchange-coupling constant J greater than 80 cm-1. The EPR spectra show non-zero rhombicity, the origin of which is discussed in terms of contributions from the usual zero-field effects of iron and from iron-radical spin-dipolar interaction. A consistent estimate of zero-field splitting parameter D approximately + 6 cm-1 was obtained by EPR and M?ssbauer measurements. EPR and M?ssbauer parameters are shown to be slightly dependent on solvent, but not on the axial ligand in the starting (TMP)Fe complex. In contrast to the TMP complex, the oxoferrylporphyrin cation radical system generated from [TPP(2,6-Cl)FeOSO2CF3] exhibits M?ssbauer and EPR spectra consistent with weak iron-porphyrin radical coupling of magnitude of J approximately 1 cm-1.     

M?ssbauer study of red blood cells from patients with erythremia

Red blood cell samples from several patients with erythremia have been studied by M?ssbauer spectroscopy. Quadrupole splitting and isomer shift for oxyhemoglobin from erythremic red blood cells and those of oxyhemoglobin from normal ones differ slightly, while these hyperfine parameters for deoxyhemoglobins are the same. An additional component in M?ssbauer spectra of red blood cell samples was observed in some cases of erythremia. It is proposed that this component is related to ferritin-like iron and its content ranged from 13-15% to 5-7%.     

Difference in the M?ssbauer spectrum parameters and electron structure of active sites of non-equivalent subunits of tetrameric deoxyhemoglobin and deoxymyoglobin

The calculations of the electronic structure of fifth-coordinated ferroporphyrin-imidazole complexes modeling alpha- and beta-subunits in desoxyhemoglobin and desoxymyoglobin are made using the interative extended Hückel method. The features of the electronic structure of the model complexes resulting from the stereochemical differences of the active site are studied and compared. Theoretical calculations of the M?ssbauer parameters of model complexes are made and compared with experimental data. The results show that basic features of the M?ssbauer parameters are qualitatively confirmed by the theoretical analysis. The necessity of the accounting of the stereochemical and electronic structure nonequivalence of nonidentical subunits in tetrameric desoxyhemoglobins during the M?ssbauer spectra approximation is also confirmed.