Spectral hole burning study of protoporphyrin IX substituted myoglobin.

Protoporphyrin IX substituted myoglobin reveals excellent hole burning properties. We investigated the frequency shift of persistent spectral holes under isotropic pressure conditions in a range from 0 to 2.4 MPa. In this range, the protein behaves like an elastic solid. The shift of the holes under pressure shows a remarkable frequency dependence from which the compressibility of the protein can be determined. The compressibility, in turn, allows for an estimation of the equilibrium volume fluctuations. Within the frame of the model used to interpret the pressure data, it is possible to determine the absorption frequency of the isolated chromophore and the associated solvent shift in the p…

The active center of superoxide dismutase from Propionibacterium shermanii

A self-consistent description of the EPR spectra and of the Mossbauer spectra of the natural superoxide dismutase from Propionibacterium shermanii with ferric iron as an active centre is presented. The spectra were measured at pH 6.5, 7.8 and 9.4. The theoretical approach is based on the use of the complete crystal field Hamiltonian for the high-spin ferric complexes with due regard for the terms of the fourth power of the electronic spin. It is shown that a SOD molecule can exist in two conformations. The low-pH conformation has predominantly trigonal symmetry, while the high-pH conformation has the symmetry close to the «extreme rhombic». This interpretation is in full agreement with EXAF…

Inter- and intramolecular motions in proteins

The use of 57 Fe Mossbauer radiation allows the study of protein crystal dynamics by a time-resolved analysis of X-ray scattering. In myoglobin crystals, the main source of the root mean squared amplitude of motions come from intramolecular protein dynamics. Segments of the size of an α-helix move collectively. Long-range correlated motions give only a minor contribution. Comparison with Mossbauer absorption spectroscopy shows that protein-specific dynamics is frozaen out below 200 K and the lattice dynamics in mainly responsible for the low-temperature behavior

An interpretation of EPR spectra of azide ligated superoxide dismutase from Propionibacterium shermanii

A self-consistent description of the EPR spectra of the azide ligated SOD is obtained by taking into account the general configuration of the crystal field splitting in the crystal field Hamiltonian. The spread in the rhombicity parameter due to the presence of different conformational substates is introduced.

Spectral broadening of the Soret band in myoglobin: an interpretation by the full spectrum of low-frequency modes from a normal modes analysis.

In this work the temperature dependence of the Soret band line shape in carbon-monoxy myoglobin is re-analyzed by using both the full correlator approach in the time domain and the frequency domain approach. The new analyses exploit the full density of vibrational states of carbon-monoxy myoglobin available from normal modes analysis, and avoid the artificial division of the entire set of vibrational modes coupled to the Soret transition into "high-frequency" and "low-frequency" subsets; the frequency domain analysis, however, makes use of the so-called short-times approximation, while the time domain one avoids it. Time domain and frequency domain analyses give very similar results, thus s…

A reduction of protein specific motions in co-ligated myoglobin embedded in a trehalose glass

Investigation of the dynamics of bacteriorhodopsin

Bacteriorhodopsin (bR) converted to the blue form by deionization has been reconstituted to the active purple membrane by addition of57Fe ions. Mossbauer spectra measured in a wide temperature range reveal Fe3+ binding places with oxygen atoms in the neighbourhood. No evidence for a well defined functional binding place of the iron has been found. On a timescale faster 100 ns the purple membrane shows increasing flexibility above 200 K. In order to analyse the influence of the lipids, a bacteriorhodopsin sample where the lipid content has been increased artificially by the incorporation of DMPC as well as a sample consisting of lipid bilayer have been investigated.

The photocycle and the structure of iron containing bacteriorhodopsin ?a kinetic and M�ssbauer spectroscopy investigation

Bacteriorhodopsin (bR), converted by deionization to the blue form was reconstituted to the active purple membrane by the addition of Fe2+ or Fe3+ ions. 57Fe Mossbauer spectra of these samples were measured at different pH values (pH 3.9, pH 5.0 and pH 7.0) and at temperatures ranging from 4 K to 300 K. The hyperfine parameters reveal two iron environments with oxygen atoms in the neighbourhood of iron. Iron type 1 is in the 3+ high spin state. It is bound to acid side chains of the protein and/or the phosphate groups of the lipids. Iron type 2 is in the 2+ high spin state and is linked to carboxy groups of the protein in a rather unspecific way. Dynamics as measured by Mossbauer spectrosco…

The Mössbauer effect and collective motions in glass-forming liquids and polymeric networks

Glass-forming liquids, synthetic polymers and biopolymers share essential properties. Dynamic processes in these complex systems are characterized by cooperative motions with wide distributions of time scales, which manifest themselves in broad quasielastic lines in the Mossbauer spectrum. In this article, the application of the Mossbauer effect to the study of structural dynamics in complex systems is discussed.

Recombination studies of photodissociated MbCO by Mössbauer spectroscopy at low temperatures

Rebinding of carbonmonoxide to myoglobin after photodissociation has been studied by Mossbauer spectroscopy at 57.6 K and below for up to 9 days. The time dependence is reproduced by a set of exponentials representing a distribution of activation enthalpies. A shift to smaller values of these activation enthalpies and of the preexponential factor compared to optical studies at higher temperatures has been observed as well as pumping into long-living states.

Dynamics of protein-water systems revealed by Rayleigh scattering of Mössbauer radiation (RSMR)

A critical review of recent studies of protein dynamics by the RSMR technique is given. The main approximations in quantitative analyses of RSMR data are discussed and conclusions about dynamical properties of protein and interprotein water, deduced from experiments, are described.

Hemoglobin dynamics in rat erythrocytes investigated by M�ssbauer spectroscopy

Rats have been enriched in 57Fe and erythrocytes were isolated from the blood. Mössbauer absorption spectroscopy on the hemoglobin of these erythrocytes has shown rather similar dynamics as found earlier in crystals of myoglobin, in frozen solutions of human hemoglobin and in a number of other proteins. The results strongly indicate that the motion of the heme and presumably some part of the F-helix is mainly influenced by the average viscosity of the sample determined by a network of hydrogen bridges and other weak interactions. Extrapolations of Mössbauer results from protein crystals to proteins in their physiological surroundings seem to be suitable for heme proteins.

Mössbauer Spectroscopy on Photosynthetic Bacteria: Investigation of Reaction Centers of Rhodopseudomonas Viridis

Crystals of 57Fe enriched reaction centers have been investigated by Mossbauer spectroscopy. The cytochrome irons are in the low spin ferric state. The non-heme iron of the electron accepting side is partly ferrous high spin and partly ferrous low spin (or ferric high spin). Under the conditions of the experiment sodium ascorbate reduces only one cytochrome iron into the ferrous low spin state. Membrane bound proteins become flexible at higher temperatures than proteins with a hydrophilic surface. They are also less flexible, at least up to temperatures of about 250 K.

Mössbauer investigations on glass-forming organic liquids

Glycerol forms a molecular glass near 180K. Fe2+ dissolved in glycerol allows the study of the dynamics of the system by Mossbauer spectroscopy. Recently it has been shown that the Mossbauer spectra can be understood in a way consistent with the results of dielectric and ultrasonic viscoelastic relaxation measurements. A jump diffusion model of Sinqwi and Sjolander with a jump rate distribution according to Davidson and Cole allowed us to fit the Mossbauer spectra of Fe in glycerol. First attempts to compare mode coupling theory with Mossbauer spectra are reported.

The hydration shell of myoglobin.

The space in the unit cell of a metmyoglobin crystal not occupied by myoglobin atoms was filled with water using Monte Carlo calculations. Independent calculations with different amounts of water have been performed. Structure factors were calculated using the water coordinates thus obtained and the known coordinates of the myoglobin atoms. A comparison with experimental structure factors showed that both the low and the high resolution regime could be well reproduced with 814 Monte Carlo water molecules per unit cell with a B-value of 50 A2. The Monte Carlo water molecules yield a smaller standard R-value (0.166) than using a homogeneous electron density for the simulation of the crystal w…

Molecular tunneling and pumping effects in low temperature MBCO recombination

Recombination of carbonmonoxide after photodissociation has been studied by Mossbauer spectroscopy at 4.2K and in the low temperature region, where tunneling effects play an important role in rebinding. We interpret the kinetic results in terms of a radiationless nonadiabatic multiphonon transition, which leads to a uniform description for all temperatures. Prolonged illumination at low temperature results in pumping into long-living states.

Structural fluctuations in glass-forming liquids: Mössbauer spectroscopy on iron in glycerol.

M\"ossbauer absorption spectra of $^{57}\mathrm{Fe}^{2+}$ dissolved in a glycerol-water mixture have been measured over a wide temperature range (80--275 K). In the supercooled liquid state the shapes of the spectra depend strongly on temperature. The M\"ossbauer spectra can be fit with jump diffusion with a Cole-Davidson distribution of fluctuation times. The results are in agreement with those obtained from dielectric and ultrasonic volume relaxations, proving that the \ensuremath{\alpha} relaxation of viscous liquids is responsible for the line broadening of the spectra. From the temperature dependence of the integrated area of the M\"ossbauer spectrum a second dynamic process is inferre…

Mössbauer spectroscopy on the reaction center of Rhodopseudomonas viridis

Proteins called “reaction centers” (RC) can be isolated from many photosynthetic bacteria. They have one non-heme iron in a quinone acceptor region. The RC of Rhodopseudomonas viridis contains an additional tightly bound tetra-heme cytochrome c subunit. The electronic configuration of both cytochrome and the non-heme iron has been studied in the crystallized protein by Mossbauer spectroscopy at different redox potentials, pH-values, and with an addition of o-phenanthroline. At high potentials (Eh=+500mV) all heme irons are in the low spin Fe3+-state, and at low potential (Eh=−150mV) they are low spin Fe2+ with the same Mossbauer parameters for all hemes independent of pH. Redox titrations c…

Temperature dependence of the dynamics of ultrafine particles in a polymeric network

Simple model systems with pronounced dynamical features will help to get a deeper insight into the complicated dynamics of large molecular networks. We investigated the bounded diffusion of ultrafine Fe(OH)3 particles (∼30 A in diameter) in the three-dimensional network of the cation exchanger Dowex 50 W which was solvated with a water solution of sucrose (60 wt%). Mossbauer spectra were recorded in the temperature range from 80 K to 305 K. At temperatures above 250 K broad diffusional lines of different widths appear in the spectrum proving the bounded nature of the diffusion. The line widths strongly increase with temperature to values of several hundred mm/s. Around 300 K a large portion…

Dielectric relaxation models applied of the dynamics of myoglobin as determined by Mössbauer spectroscopy

Abstract Protein specific modes of motions are found in myoglobin crystals above 180 K. In this contribution we show that this type of motions can be analyzed by a Davidson-Cole, a Cole-Cole or a Havriliak-Negami distribution in analogy to dielectric relaxation. However, the temperature dependence of the obtained parameters is quite unusual indicating a broadening of the distributions with temperature instead of motional narrowing. This can be understood from the picture of conformational substates if one assumes that more and more substates become accessible with increasing temperature. The result shows that the analogy between glass forming organic liquids and proteins should not be exagg…

X-ray structure determination of a metastable state of carbonmonoxy myoglobin after photodissociation.

The x-ray structure of carbon monoxide (CO)-ligated myoglobin illuminated during data collection by a laser diode at the wavelength lambda = 690 nm has been determined to a resolution of 1.7 A at T = 36 K. For comparison, we also measured data sets of deoxymyoglobin and CO-ligated myoglobin. In the photon-induced structure the electron density associated with the CO ligand can be described by a tube extending from the iron into the heme pocket over more than 4 A. This density can be interpreted by two discrete positions of the CO molecule. One is close to the heme iron and can be identified to be bound CO. In the second, the CO is dissociated from the heme iron and lies on top of pyrrole ri…

Rayleigh scattering of Mössbauer radiation on a myoglobin single crystal

The RSMR technique was used to determine the fraction of radiation which is scattered elastically and inelastically, respectively, into Bragg reflections of a metmyoglobin single crystal. From this measurement 〈x2 1c〉=0.027±0.008 A2 has been determined which is due to long range correlated motions in analogy to acoustic and optic modes in solids.