Structural and dynamic properties of the heme pocket in myoglobin probed by optical spectroscopy

We report the optical absorption spectra of sperm whale deoxy-, oxy-, and carbonmonoxymyoglobin in the temperature range 300–20 K and in 65% glycerol or ethylene glycol–water mixtures. By lowering the temperature, all bands exhibit half-width narrowing and peak frequency shift; moreover, the near-ir bands of deoxymyoglobin show a marked increase of the integrated intensities. Opposed to what has already been reported for human hemoglobin, the temperature dependence of the first moment of the investigated bands does not follow the behavior predicted by the harmonic Franck–Condon approximation and is sizably affected by the solvent composition; this solvent effect is larger in liganded than in nonliganded myoglobin. However, for all the observed bands the behavior of the second moment can be quite well rationalized in terms of the harmonic Franck–Condon approximation and is not dependent on solvent composition. On the basis of these data we put forward some suggestions concerning the structural and dynamic properties of the heme pocket in myoglobin and their dependence upon solvent composition. We also discuss the different behaviors of myoglobin and hemoglobin in terms of the different heme pocket structures and deformabilities of the two proteins.     

Mössbauer spectroscopic studies of hemoglobin and its isolated subunits.

Samples of 90% enriched 57Fe hemoglobin and its isolated subunits have been prepared. M?ssbauer spectroscopic measurements have been made on three such samples. Sample one contained contributions of oxyhemoglobin, deoxyhemoglobin, and carbonmonoxyhemoglobin. This sample was studied from a temperature of 90 K down to 230 mK. Measurements were also made at 4.2 K using a small applied magnetic field of 1.0 T. In general, the measured quadrupole splittings and isomer shifts for each component agreed with previous measurements on single component samples in the literature, and thus demonstrated that chemically enriched hemoglobin has not been altered. The second and third samples were isolated alpha and beta subunits, respectively. We have found measurable M?ssbauer spectral differences between the HbO2 sites in the alpha subunit sample and the beta subunit sample. The measured M?ssbauer spectral areas indicate that the iron ion has the largest mean-square displacement at the deoxy Hb sites as compared to that at the oxy- and carbonmonoxy Hb sites. The mean-square displacement at the HbO2 sites is the smallest.     

Dynamics of hemoglobin investigated by M?ssbauer spectroscopy

M?ssbauer Spectra of Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-M?ssbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-M?ssbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solution above 200 K where the Lamb-M?ssbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.     

Induced optical activity in the complex of poly(L-arginine) with azo dyes

The absorption and CD spectra of the complexes of poly(L -arginine) (PLA) and azo dyes have been measured in aqueous solution. On complexation, Blue-shifted additional absorption bands were observed. In the wide pH 4–11 range, induced CD was observed at the visible wavelengths corresponding to the blue-shifted absorption bands. The induced CD arose from the dimeric dye molecules bound to PLA in the α-helical structure. When a modified analysis of induced CE is made by the excition chirality method, the origin of the induced CD can be assigned to the dipole coupling. The PLA–dye complexes showed the counterlockwise (negative, S) chirality of the transition dipole moments of dyes.     

Dynamics of Hemoglobin Investigated by Mössbauer Spectroscopy

Abstract

Mössbauer Spectra of ⁵⁷Fe enriched horse hemoglobin and sperm whale myoglobin were measured in the temperature range from 80 K to 260 K. An analysis of the temperature dependence of the recoiless fraction (the Lamb-Mössbauer factor) shows it to be sensitive to conformational fluctuations which affect the mean square displacement of the iron. We have found that the protein conformation has a dramatic effect on these measurements. For hemoglobin greater conformational fluctuations at lower temperatures are observed for carbonmonoxyhemoglobin in the liganded conformation than for deoxyhemoglobin in the unliganded conformation. On the other hand, the Lamb-Mössbauer factor is insensitive to the binding of ligands to myoglobin and shows conformational fluctuations similar to deoxyhemoglobin even in the liganded state. It is also shown that a reversible complex with the distal histidine is formed in frozen deoxyhemoglobin solutions above 200 K where the Lamb-Mössbauer factor shows the excitation of new modes of conformational fluctuations. This complex is not formed with carbonmonoxyhemoglobin which already has a sixth ligand and with deoxymyoglobin which appears to undergo much more limited conformational fluctuations. A possible relationship between the formation of the distal histidine complex and the cooperative ligand binding reaction is suggested by results with partially liganded hemoglobin which indicate increased formation of the distal histidine complex.     

Variation in ultraviolet reflectance by carotenoid-bearing feathers of tanagers (Thraupini: Emberizinae: Passeriformes)

Avian visual sensitivity encompasses both the human visible range (400–700 nm) and also near‐ultraviolet (UV) wavelengths (320–400 nm) invisible to normal humans. I used reflectance spectrophotometry to assess variation in UV reflectance for yellow, orange and red plumage in 67 species of tanager (Passeriformes). Previous chemical studies, and my analysis of reflectance minima, suggest that carotenoids are the dominant pigments in yellow, orange and red tanager plumage. Spectra recorded over the range of wavelengths to which birds are sensitive (320–700 nm) were invariably bimodal, with both a plateau of high reflectance at longer (> 500 nm) wavelengths and a distinct secondary peak at UV (< 400 nm) wavelengths. Within this overall framework, variation in UV reflectance was expressed within well‐defined quantitative limits: (1) peak reflectance was always lower than the corresponding plateau of reflectance at longer visible wavelengths; (2) the intensity of peak reflectance declined steadily below 350 nm; (3) wavelengths of peak reflectance clustered between 350 and 370 nm. Significant correlations were detected between various measures of total reflectance in the UV and visible wavebands, but not between various measures of spectral location of UV and visible reflectance. I propose that the strong absorption band at short visible wavelengths (～ 380–550 nm) responsible for bimodal spectra of carotenoids in vitro is also responsible for bimodal reflectance by carotenoid‐based plumage colours. The construction of the UV and visible reflectance bands from different sides of this same absorbance band provides a mechanism for the observed covariation between UV and visible wavelengths. Lack of an association between the spectral locations of the UV and visible reflectance bands may result from the limited variation in spectral location of the UV band. These patterns suggest that plumage colours are subject to constraints, just as are more traditional morphological characters. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 84 , 243–257.     

High pressure NMR studies of hemoproteins. The effect of pressure on the tertiary and quaternary structures of human adult ferrous hemoglobin

The effect of pressure on the tertiary and quaternary structures of human oxy, carbonmonoxy, and deoxyhemoglobin was examined by high pressure NMR spectroscopy at 300 MHz. The increased pressure displaced the ring current-shifted gamma 1-methyl resonance of beta E11 valine for oxy- and carbonmonoxyhemoglobin to the upfield side, whereas that of the alpha subunit was insensitive to pressure. Such a preferential pressure-induced upfield shift for the beta E11 valine gamma 1-methyl signal was also encountered for the isolated carbonmonoxy beta chain. For deoxyhemoglobin, hyperfine shifted resonances of the heme peripheral proton groups and the proximal histidyl NH proton for the beta subunit were pressure-dependent, in contrast to the pressure-insensitive responses for these resonances of the alpha subunit. These results indicate the structural nonequivalence of the pressure-induced structural changes in the alpha and beta subunits of hemoglobin. The exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds which have been used as the oxy and deoxy quaternary structural probes were not changed upon pressurization. From all of above results, it was concluded that pressure induces the tertiary structural change preferentially at the beta heme pocket of the ferrous hemoglobin derivatives with the quaternary structure retained.     

The altitudinal gradient of vascular plant richness in Aurland, western Norway

Differences in vascular plant species richness; along the altitudinal gradient in the Aurland area of western Norway have been investigated. Based on field surveys, as complete lists as possible of all vascular plants have been compiled for each 100 m altitudinal band, from sea level to the highest mountain (1764 m). For each of the 18 altitudinal bands, climatic data have been estimated. A total of 444 vascular plant species were recorded. Highest species richness (263 species) occurred in the 600–700 in band, whereas the uppermost band had only 10 species. There are minor differences in species number between the altitudinal bands < 1000 m. Partial least squares regression shows that species richness for the overall altitudinal gradient is well predicted by mean July and January temperatures and mean annual precipitation. Species turnover is highest in the 100–200 m. 600–700 m. and 1400–1500 m altitudinal bands. In terms of the gradient in summer temperature, the study supports the generally assumed linear relationship between July temperature and the number of vascular plant species between 700 and 1500 m corresponding with a mean July temperature range of 7–11°C. The study shows a decrease of ca 30 vascular plant species with a 1°C decrease in mean July temperature, and that the “climatic vascular plant limit” is here estimated to occur at a mean July temperature of 2.4°C. Above 1500 and below 700 m. species number is lower than expected based on summer iemperature conditions alone. The 700–800 m band represents the highest floristic difference compared to the other bands. Ordination and classification analyses of the floristic compositional data of all the bands highlight the 600–800 and 1500–1600 m altitudinal bands as the major biotic boundaries along the gradient. No major discontinuity in species richness, composition, or turnover was consistently found, however, at the 1100–1200 m band representing the forest-limit ecotone in Aurland.     

Linking physiological parameters with visible/near-infrared leaf reflectance in the incubation period of vascular wilt disease

The photosynthetic pigments are mainly responsible for absorbing the light intended to promote photosynthesis on the chloroplast of the leaves. Different studies have related the spectral response in the leaves of plants with the biotic stress generated by pathogens. In general, maximum differences in reflectance have been found in the range of 380–750 nm between plants subjected to biotic stress and healthy plants. In this study, it was possible to characterize and relate the spectral variance in leaves of S. lycopersicum infected with F. oxysporum with this physiological variation and pathogen concentration in tomato plants during the asymptomatic period of vascular wilt. Photosynthetic parameters derived from gaseous exchange analysis in the tomato leaves correlated related with four bands in the visible range (Vis). Additionally, five specific bands also present a high correlation with the increase in the concentration of F. oxysporum conidia measured at the root: 448–523 nm, 624–696 nm, 740–960 nm, 973–976 nm, and 992–995 nm. These wavelengths allowed a 100% correct classification of the plants inoculated with F. oxysporum from the plants subjected to hydric stress and the control plants in the asymptomatic period of the disease. The spectral response to biotic and abiotic stress in the measured Vis/NIR range can be explained by the general tendency to change the concentration of chlorophyll and carotene in tomato leaves. These studies also highlight the importance of the implementation of robust multivariate analysis over the multiple univariate analysis used in the applied biological sciences and specifically in the agricultural sciences. These results demonstrate that specific wavelength responses are due to physiological changes in plants subjected to stress, and can be used in indexes and algorithms applied to the early detection of diseases in plants on different pathosystems.     

The binding of CO2 to human hemoglobin.

CO2-dissociation curves of concentrated human deoxy- and carbonmonoxyhemoglobin at 37 degrees, pH 7.6 to 7.0, PCO2 equal to 10 to 160 mm Hg, have been obtained by a rapid mixing and ion exchange technique. The CO2-dissociation curves for deoxyhemogloblin can only be fitted by assuming two classes of binding sites for carbon dioxide. The simplest way to account for the experimental data is to assume that the alpha-amino groups of the alpha and beta chains react with carbon dioxide with affinities that differ by at least a factor of 3. No difference in reactivity with CO2 was found among the four terminal alpha-amino groups of carbonmonoxyhemoglobin.     

Effects of hydration in the oxygenation and autoxidation of carbonmonoxyhemoglobin powder

The oxygenation and autoxidation of lyophilized human carbonmonoxyhemoglobin (Hb-CO) kept in environments with different relative humidities were followed with time using visible absorption spectroscopy. The sample kept at 68% relative humidity was the one with the greatest formation of methemoglobin (Met-Hb), while for increasing or decreasing hydrations the Met-Hb content decreased gradually. Besides the presence of Met-Hb, it was observed that the samples kept above about 68% relative humidity were converted to oxyhemoglobin (Hb-O2), the samples kept in the range 45-68% relative humidity were a mixture of Hb-CO and Hb-O2, while the samples kept below about 45% relative humidity and the solution sample continued as Hb-CO. Hemoglobin has, therefore, two critical hydration values, 45% and 68% relative humidity, which correspond to about 0.12 and 0.18 g H2O/g protein, respectively.     

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.     

A new spectrophotometric cuvette holder for low temperature studies; Its application to the study of carbonmonoxyhemoglobin oxidation rate

A new spectrophotometric cuvette holder to be used for subzero temperature is described. The device is easily adaptable to a commercial spectrophotometer and it was checked down to --40 degrees C. Satisfactory mixing of the reactants contained in the cuvette at low temperatures is attained using a special stirrer and suitable solution volumes. The rate of carbonmonoxyhemoglobin oxidation by K3Fe(CN)6 at different subzero temperatures has been studied using this apparatus; the results are in agreement with the extrapolated data at room temperature.     

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.     

Preparative isoelectric focusing of CO hemoglobins on polyacrylamide gels and conversion to their oxy forms

Isoelectric focusing on acrylamide gels has been used to prepare 100–200 mg amounts of modified hemoglobins in a state of high purity. A method for the quantitative elution of the protein from the gel is described. A new procedure for the complete conversion of carbonmonoxyhemoglobin derivatives to their oxy or deoxy forms which fully preserves their functional integrity has also been developed.     

Preparation and analysis of small unilamellar phospholipid vesicles of a uniform size

The interaction of carbonmonoxyhemoglobin and heme with small unilamellar phospholipid vesicles was studied using dynamic light scattering. Addition of carbonmonoxyhemoglobin to dimyristoylphosphatidylcholine:dimyristoylphosphatidylserine small unilamellar vesicles resulted in an increase of average vesicle size from 17.4 to 32.0nm. Addition of heme to vesicles produced a smaller size increase, from 17.4 to 21.0nm. Also reported is a method for preparing small unilamellar lipid vesicles of a uniform size, suitable for use in NMR spectroscopy.     

Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Influence of absorption on polarization effects in light scattering from human red blood cells

Polarization effects in light scattering are sensitive indicators of cell structure and structural changes in time. In the spectral regions where the optical properties of the scatterers are relatively constant, the scattering pattern scales, it contracts or expands in a predictable manner as a function of the wavelength. In the spectral regions where the optical properties are strongly wavelength dependent (near absorption bands, etc.) the scattering curves do not scale, but change drastically in phase and amplitude as the wavelength is varied. Reported here is an empirical study of the magnitude of the influence of absorption on the polarization effects in light scattering. Scattering curves have been obtained for human red blood cells in the absorption band (blue light) and far from the absorption band (red light). The scattering at these wavelengths shows very strong nonscaling differences. These observations suggest the use of polarization effects in light scattering and their wavelength dependence for the studies of structural changes in cell nuclei. Nucleoproteins have strong absorption, optical rotatory dispersion and circular dichroism bands in the ultraviolet region of the spectrum, whereas there is little ψ-dependence in the visible range. There is also the possibility of binding specific chromophoric dyes to cell components, thus introducing absorption bands in the visible range, where scattering instrumentation and laser light sources are more readily available.     

Influence of globin structure on the heme in dromedary carbonmonoxyhemoglobin

By use of X-ray absorption near-edge structure (XANES), circular dichroism, and visible absorption spectroscopies, dromedary carbonmonoxyhemoglobin has been characterized structurally and functionally. By consideration of the experimental results the following view emerges: (i) the quaternary structure is not the unique factor determining the tertiary environment around the heme, and (ii) the multiplicity of interactions between hemoglobin and solvent components induces a large number of globin conformations, which somehow affect the conformation of the heme such that the structural parameters (i.e., the doming of porphyrins, the movements of the iron relative to the heme plane, the distortion of the ligand field, and the change in the Fe-C-O angle) can be uncoupled.     

Equations for the spectrophotometric analysis of hemoglobin mixtures

The extinction coefficients for deoxyhemoglobin, oxyhemoglobin, and carbonmonoxyhemoglobin as well as those of ferrihemoglobin between pH 6.2 and 8.8 are given at a number of wavelengths. Equations are presented for the analysis of ternary mixtures of these hemoglobin derivatives.