Eugenio Vitrano

Optical absorption spectra of deoxy- and oxyhemoglobin in the temperature range 300-20 K: relation with protein dynamics

We have studied the optical absorption spectra of human deoxy- and oxyhemoglobin in the temperature range 300-20 K and in the wavelength range 350-1350 nm. By lowering the temperature, a narrowing and a shift of all bands were observed together with a sizeable increase of the integrated intensities of the charge-transfer bands of deoxyhemoglobin. At all temperatures the spectra are in full agreement with the band assignment previously suggested in the literature and no new relevant bands have been detected for both deoxy- and oxyhemoglobin. Analysis of the first and second moment of the bands, within the framework of the harmonic Franck-Condon approximation, gave information on the dynamic properties of the heme in the heme pocket.

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.

Conformational and functional properties of haemoglobin in perturbed solvent: relevance of electrostatic and hydrophobic interactions

Abstract We present a review of a series of papers, worked out at our laboratory, showing how the aqueous solvent medium regulates the conformational and functional properties of haemoglobin. In particular, in these papers, we succeeded in providing evidence of the relevance of two contributions: one, electrostatic in nature, related to the value of the bulk dielectric constant of the medium, the other related to protein solvent hydrophobic interactions.

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.

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.

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.

Dynamic properties of the active site of azurin studied by the temperature dependence of the optical spectrum

SummaryWe report the optical absorption spectra of azurin (Pseudomonas aeruginosa) in the temperature range 290-20 K. The samples used are protein aqueous solutions containing 65% (by Vol.) glycerol as cryoprotectant. The measured spectra are deconvoluted in gaussian components and the temperature dependence of the zeroth, first and second moment of the observed bands is analyzed using the harmonic Franck-Condon approximation for the coupling between electronic transitions and nuclear vibrations. The analysis provides information on the stereodynamic properties of the active site of this protein. The possible functional relevance of these results is also suggested.

The interaction of sodium maleate with human haemoglobin.

We have studied the effect of sodium maleate on the reaction of haemoglobin with oxygen at various temperatures between 10 degrees C and 30 degrees C. At all the temperatures investigated, the presence of sodium maleate causes a significant increase of log p50. Analysis of the experimental data in terms of Wymans linkage theory indicates that one oxygen-linked maleate molecule bound to the haemoglobin tetramer in the deoxy conformation is released when the tetramer is oxygenated. Further analysis within the framework of the Monod-Wyman-Changeux model, considering maleate as a classical allosteric inhibitor, enabled us to obtain the equilibrium constant for the binding of maleate to deoxyhaemoglobin at various temperatures and therefore the values of the standard binding entropy and enthalpy. These last values indicate that the binding of maleate to deoxyhaemoglobin is an exothermic enthalpy-driven process.