Dehydration and crystallization of trehalose and sucrose glasses containing carbonmonoxy-myoglobin.

We report a study wherein we contemporarily measured 1) the dehydration process of trehalose or sucrose glasses embedding carbonmonoxy-myoglobin (MbCO) and 2) the evolution of the A substates in saccharide-coated MbCO. Our results indicate that microcrystallization processes, sizeably different in the two saccharides, take place during dehydration; moreover, the microcrystalline structure is maintained unless the dry samples are equilibrated with a humidity >/=75% (>/=60%) at 25 degrees C for the trehalose (sucrose) sample. The evolution of the parameters that characterize the A substates of MbCO indicates that 1) the effects of water withdrawal are analogous in samples dried in the presence or in the absence of sugars, although much larger effects are observed in the samples without sugar; 2) the distribution of A substates is determined by the overall matrix structure and not only by the sample water content; and 3) the population of A0 substate (i. e., the substate currently put in relation with MbCO molecules having the distal histidine out of the heme pocket) is largely enhanced during the dehydration process. However, after rehumidification its population is largely decreased with respect to the values obtained, at similar water content, during the first dehydration run.

Harmonic behavior of trehalose-coated carbon-monoxy-myoglobin at high temperature.

Embedding biostructures in saccharide glasses protects them against extreme dehydration and/or exposure to very high temperature. Among the saccharides, trehalose appears to be the most effective bioprotectant. In this paper we report on the low-frequency dynamics of carbon monoxy myoglobin in an extremely dry trehalose glass measured by neutron spectroscopy. Under these conditions, the mean square displacements and the density of state function are those of a harmonic solid, up to room temperature, in contrast to D2O-hydrated myoglobin, in which a dynamical transition to a nonharmonic regime has been observed at approximately 180 K (Doster et al., 1989. Nature. 337:754-756). The protective effect of trehalose is correlated, therefore, with a trapping of the protein in a harmonic potential, even at relatively high temperature.

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide.

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the proximity of the chromophore. For the dye free in solution, large spectral heterogeneity and coupling to a "bath" of low-frequency (soft) modes is observed; moreover, anharmonic motions become evident at suitably high temperatures. The average frequency of the soft modes and the amplitude of anharmonic motions depend upon solvent composition. For the DNA-bound dye, at low temperatures, heterogeneity is decreased, the average frequency of the soft modes is increased, and anharmonic motions are hindered. However, a new dynamic regime characterized by a large increase in anharmonic motions is observed at temperatures higher than approximately 280 K. The DNA double helix therefore appears to provide, at low temperatures, a rather rigid environment for the bound chromophore, in which conformational heterogeneity is reduced and low-frequency motions (both harmonic vibrations and anharmonic contributions) are hindered. The system becomes anharmonic at approximately 180 K; however, above approximately 280 K, anharmonicity starts to increase much more rapidly than for the dye free in solution; this can be attributed to the onset of wobbling of the dye in its intercalation site, which is likely connected with the onset of (functionally relevant) DNA motions, involving local opening/unwinding of the double helix. As shown by parallel measurements of the melting curves, these motions precede the melting of the double helix and depend upon solvent composition much more than does the melting itself.

Heme geometry in the 10 K photoproduct from sperm whale carbonmonoxymyoglobin.

We have measured the Soret band of the photoproduct obtained by complete photolysis of sperm whale carbonmonoxymyoglobin at 10 K. The experimental spectrum has been modeled with an analytical expression that takes into account the homogeneous bandwidth, the coupling of the electronic transition with both high and low frequency vibrational modes, and the effects of static conformational heterogeneity. The comparison with deoxymyoglobin at low temperature reveals three main differences. In the photoproduct, the Soret band is shifted to red. The band is less asymmetric, and an enhanced coupling to the heme vibrational mode at 674 cm-1 is observed. These differences reflect incomplete relaxation of the active site after ligand dissociation. The smaller band asymmetry of the photoproduct can be explained by a smaller displacement of the iron atom from the mean porphyrin plane, in quantitative agreement with the X-ray structure analysis. The enhanced vibrational coupling is attributed to a subtle heme distortion from the planar geometry that is barely detectable in the X-ray structure.

Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins.

In this short review we show how suitable analysis of the temperature dependence of the optical absorption spectra of metalloproteins can give insight into their stereodynamic properties in the region of the chromophore. To this end, the theory of coupling between an intense allowed electronic transition of a chromophore and Franck-Condon active vibrations of the nearby atoms is applied to the Soret band of hemeproteins to obtain an analytical expression suitable for fitting the spectral profile at various temperatures. The reported approach enables one to separate the various contributions to the overall bandwidth together with the parameters that characterize the vibrational coupling. The thermal behavior of these quantities gives information on the dynamic properties of the active site and on their dependence upon protein structure and ligation state. The Soret band of hemeproteins appears to be coupled to high frequency vibrational modes of the heme group (as already shown by resonance Raman spectroscopy) and to a "bath" of low frequency modes most likely deriving from the bulk of the protein. For the deoxy derivatives inhomogeneous broadening arising from conformational heterogeneity appears to contribute substantially to the linewidth. The data indicate the onset, at temperatures near 180 K, of large scale anharmonic motions that can be attributed to jumping among different conformational substates of the protein.

Stereodynamic properties of the cooperative homodimeric Scapharca inaequivalvis hemoglobin studied through optical absorption spectroscopy and ligand rebinding kinetics.

The study of the thermal evolution of the Soret band in heme proteins has proved to be a useful tool to understand their stereodynamic properties; moreover, it enables one to relate protein matrix fluctuations and functional behavior when carried out in combination with kinetic experiments on carbon monoxide rebinding after flash photolysis. In this work, we report the thermal evolution of the Soret band of deoxy, carbonmonoxy, and nitric oxide derivatives of the cooperative homodimeric Scapharca inaequivalvis hemoglobin in the temperature range 10-300 K and the carbon monoxide rebinding kinetics after flash photolysis in the temperature range 60-200 K. The two sets of results indicate that Scapharca hemoglobin has a very rigid protein structure compared with other hemeproteins. This feature is brought out i) by the absence of nonharmonic contributions to the soft modes coupled to the Soret band in the liganded derivatives, and ii) by the almost "in plane" position of the iron atom in the photoproduct obtained approximately 10(-8) s after dissociating the bound carbon monoxide molecule at 15 K.

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions.

In this work we report the thermal behavior (10-300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in turn, give information on structural and dynamic properties of the systems studied. The optical spectroscopy data point out sizable differences between elephant myoglobin on one hand and horse and sperm whale myoglobins on the other. These differences, more pronounced in deoxy derivatives, involve both the structure and dynamics of the heme pocket; in particular, elephant myoglobin appears to be characterized by larger anharmonic contributions to soft modes than the other two proteins. Flash photolysis data are analyzed as sums of kinetic processes with temperature-dependent fractional amplitudes, characterized by discrete pre-exponentials and either discrete or distributed activation enthalpies. In the whole temperature range investigated the behavior of elephant myoglobin appears to be more complex than that of horse and sperm whale myoglobins, which is in agreement with the increased anharmonic contributions to soft modes found in the former protein. Thus, to satisfactorily fit the time courses for CO recombination to elephant myoglobin five distinct processes are needed, only one of which is populated over the whole temperature range investigated. The remarkable convergence and complementarity between optical spectroscopy and flash photolysis data confirms the utility of combining these two experimental techniques in order to gain new and deeper insights into the functional relevance of protein fluctuations.

Protein dynamics: conformational disorder, vibrational coupling and anharmonicity in deoxy-hemoglobin and myoglobin.

In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300-20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0-0 electronic transition frequencies. The reported analysis enables us to isolate the various contributions to the overall bandwidth, and their temperature dependence points out the relevance of low frequency vibrations and of large scale anharmonic motions starting at temperatures higher than 170 K. Information on the mean iron-heme plane distance and on its temperature dependence, as well as on the heme pocket conformational disorder, is also obtained.

Protein dynamics. Vibrational coupling, spectral broadening mechanisms, and anharmonicity effects in carbonmonoxy heme proteins studied by the temperature dependence of the Soret band lineshape.

In this work we study the temperature dependence of the Soret band lineshape of the carbonmonoxy derivatives of sperm whale myoglobin, human hemoglobin, and its isolated alpha and beta subunits. To fit the observed spectral profile we use an analytic expression derived for a system whereby a single electronic transition is coupled to Franck-Condon active vibrational modes, within the adiabatic and harmonic approximation. The vibronic structure of the spectra arises from the coupling with high frequency modes; these modes contribute to the total line shape through a series of Lorentzians with peak positions at vibrational overtones and half width related to the time constant of the population decay of the excited electronic state (homogeneous broadening); moreover, the coupling with low frequency modes broadens each Lorentzian to a Voigtian. Inhomogeneous broadening is modeled as a gaussian distribution of the 0-0 transition frequencies and is therefore added as a constant term to the previous gaussian width. This spectral deconvolution enables us to investigate the different contributions to line broadening and the parameters that characterize the vibrational coupling, as well as their dependence upon protein and solvent composition. The investigation is carried out as a function of temperature in the range 20-300 K; relevant information is obtained by comparing experimental results with theoretical predictions. This work supports a description of the investigated proteins as heterogeneous systems, whose heterogeneity depends on the particular protein and on the composition of the external matrix. The delocalized pi electron cloud of the porphyrin ring is coupled not only to the high frequency vibrational modes of the active site but also to a "bath" of lower frequency modes that involve the entire protein; moreover at suitable temperatures (approximately 200 K), anharmonic motions, which are an obvious prerequisite for the jumping among different conformational substates, become evident.

Strong vibronic coupling in heme proteins.

We report the near infrared absorption spectra of cyanomethemoglobin and cyanometmyoglobin in two different solvents (deuterated solutions containing 65% v/v glycerol(OD)3 or 65% v/v ethylene glycol(OD)2). At 25 K the spectra show a clearly resolved fine structure that can be accounted for by considering a strong coupling of the porphyrin-to-iron charge transfer transitions with a single vibrational mode at 365 cm-1. The coupling constants depend on both the specific electronic transition and the protein surrounding the chromophore, indicating once more the specificity of heme globin interactions.

Protein dynamics. Comparative investigation on heme-proteins with different physiological roles.

We report the low temperature carbon monoxide recombination kinetics after photolysis and the temperature dependence of the visible absorption spectra of the isolated alpha SH-CO and beta SH-CO subunits from human hemoglobin A in ethylene glycol/water and in glycerol/water mixtures. Kinetic measurements on sperm whale (Physeter catodon) myoglobin and previously published optical spectroscopy data on the latter protein and on human hemoglobin A, in both solvents, (Cordone, L., A. Cupane, M. Leone, E. Vitrano, and D. Bulone. 1988. J. Mol. Biol. 199:312-218) are taken as reference. Low temperature flash photolysis data are analyzed within the multiple substates model proposed by Frauenfelder and co-workers (Austin, R. H., K. W. Beeson, L. Eisenstein, H. Frauenfelder, and I. C. Gunsalus. 1975. Biochemistry. 14:5355-5373). Within this model a distribution of activation enthalpies for ligand binding accounts for the structural heterogeneity of the protein, while the preexponential factor, containing also the entropic contribution to the free energy of the process, is considered to be constant for all conformational substates. Optical spectra are deconvoluted in gaussian components and the temperature dependence of the moments of the resulting bands is analyzed, within the harmonic Frank-Condon approximation, to obtain information on the stereodynamic properties of the heme pocket. The kinetic and spectral parameters thus obtained are found to be protein dependent also with respect to their sensitivity to changes in the composition of the external medium. A close correlation between the kinetic and spectral features is observed for the proteins examined under all experimental conditions studied. The results reported are discussed in terms of differences in the heme pocket structure and in the conformational heterogeneity among the various proteins, as related to their different capability to accommodate constraints imposed by the external medium.

The effect of organic cosolvents on the oxygen affinity of fetal hemoglobin. Relevance of protein-solvent interactions to the functional properties.

We have studied the effects of organic cosolvents (monohydric alcohols and formamide) on the oxygen affinity of human fetal hemoglobin stripped of phosphates and have compared them with the effects of the same cosolvents on the oxygen affinity of human adult hemoglobin under the same experimental conditions. Our results confirm that, in fetal hemoglobin, the T in equilibrium R conformational equilibrium is more displaced toward the T conformation than in the adult form and indicate that increased electrostatic and hydrophobic protein-solvent interactions contribute to this effect. The data reported are discussed in terms of the known amino acid substitutions between the beta- and gamma-chains and an attempt is made to rationalize the results with a molecular mechanism based on the crystallographic structure of fetal deoxyhemoglobin.

Optical absorption spectra of azurin and stellacyanin in glycerol/water and ethylene glycol/water solutions in the temperature range 290-20 K.

We have measured the optical absorption spectra of azurin and stellacyanin in the wavelength range 1100-350 nm and in the temperature interval 290-20 K. Samples are protein aqueous solutions containing 65% (v/v) glycerol or ethylene glycol as cryoprotectants and remain homogeneous and transparent throughout the whole temperature range investigated. Spectra are deconvoluted into Gaussian components and the temperature dependence of the zeroth, first and second moments of the various bands is analyzed, within the harmonic Franck-Condon approximation, to obtain information on the stereodynamic properties of the active sites of these proteins. Sizable differences of the integrated intensities of all the bands with temperature are observed and are attributed to variations of the metal-ligand relative positions (i.e., deformations of the active site) that occur as the temperature is lowered. The mean effective frequency of the nuclear vibrations coupled to all the observed bands is about 150 cm(-1) for both proteins in both solvents used; this indicates that the electronic transitions from which the optical spectrum originates are substantially coupled with low-frequency vibrational modes, likely ligand-metal-ligand deformations. The relevance of the stereodynamic properties of azurin and stellacyanin, investigated in this work, to their functional behavior is also suggested.

Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model.

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.

Interaction between external medium and haem pocket in myoglobin probed by low-temperature optical spectroscopy.

The visible absorption spectra of carbonmonoxymyoglobin in the temperature range 300 to 20 K are reported and compared with the analogous spectra of carbonomonoxyhaemoglobin. The temperature dependence of the zeroth, first and second moment of the observed bands is analysed to obtain information on the local dynamics in the proximity of the haem. Contrary to haemoglobin, the first moment of the observed bands in myoglobin is markedly affected by the solvent composition and its value saturates at temperatures at which the solvent undergoes the glass transition. These data indicate that solvent properties influence the haem pocket stereodynamics in myoglobin; moreover, the different behaviour between myoglobin and haemoglobin suggests that the process should involve the surfaces that are buried in the haemoglobin tetramer and exposed to the solvent in myoglobin, and/or the different protein compressibility.