Dynamic properties of oxy- and carbonmonoxyhemoglobin probed by optical spectroscopy in the temperature range of 300-20 K

We have studied the absorption bands of oxy- and carbonmonoxyhemoglobin in the wavelength range of 650–350 nm (visible and Soret bands) and in the temperature range of 300–20 K. The spectra in the whole temperature range have been successfully deconvoluted in terms of gaussian components. The analysis of the temperature dependence of the first and second moment of the bands enables us to compare dynamic properties of the heme group in oxy- and carbonmonoxyhemoglobin. The results of the analysis indicate that the “mean-effective” frequency of the nuclear motion coupled to the electronic transition responsible for the visible bands is higher in carbonmonoxy- than in oxyhemoglobin. The possible functional relevance of this finding is discussed.     

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.     

Comparative spectral absorption characteristics of hemoglobins in elasmobranches,cartilaginous ganoids,and Teleostei

The spectrophotometric parameters of the main hemoglobin forms, deoxy-, oxy-, and carboxyhemoglobin, were studied in representatives of cartilaginous ganoids (giant sturgeon), elasmobranches (piked dogfish, thresher), and bony fish (the Mediterranean horse mackerel). It was established that the hemoglobin absorption spectra in the studied fish species do not have distinct specific features with respect to positions of maxima of absorption bands, their number, and extinctions. There were differences revealed in the process of deoxygenation of oxyhemoglobin in cartilaginous fish, on one hand, and cartilaginous ganoids and bony fish, on the other hand. Incomplete deoxygenation of the piked dogfish oxyhemoglobin appears to be due to the presence of two hemoglobin fractions with different values of the Bohr effect.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 29–32.Original Russian Text Copyright © 2005 by Vasilev.     

pH-dependent absorption in the B and Q bands of oxyhemoglobin and chemically modified oxyhemoglobin (BME) at low Cl- concentrations.

We have measured the optical absorbance in the maxima of the Q and B bands for oxyhemoglobin and oxyhemoglobin (BME) in dependence on the pH value of the solution in the region between pH 4.4 and pH 10. From the absorbance data optical titration curves are derived for both bands. These yield for oxyhemoglobin pK values 4.3, 5.3, 6.8, 7.8, and 9.0, whereas for oxyhemoglobin (BME) only one pK value at 4.3 is observed. These data are in good agreement to those derived recently from resonance Raman spectroscopy. The changes of the oscillator strengths in the Q bands are interpreted in terms of Gouterman's four-orbital model to arise from A1g-distortions of the heme group, resulting from changes of the heme-apoprotein interactions due to protonation processes of amino acid-side groups in the beta-chains. The difference between the sets of pK values in oxyhemoglobin and oxyhemoglobin BME is explained from the fact that the bifunctional reagent BME blocks important pathways of heme-apoprotein interactions. The fact that in any case increase of the Q band absorbance is accompanied by a corresponding increase in the B band absorbance leads us to the conclusion that the electronic structure of the B bands has to be described in terms of a six-orbital model, taking into account configurational interaction with the L and N bands.     

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.Abbreviations Cc Carbon monoxide - Hb Human deoxyhemoglobin A - HbCO human carbonmonoxyhemoglobin A - SWMb spermwhale deoxymyoglobin - SWMbCO spermwhale carbonmonoxymyoglobin - HbO₂ human oxyhemoglobin A - SWMb³⁺-H₂O spermwhale aquometmyoglobin     

Resonance Raman spectra of bovine liver catalase: enhancement of proximal tyrosinate vibrations

Resonance Raman spectra of native bovine liver ferri-catalase have been obtained in the 200-1800 cm-1 region. Excitation at a series of wavelengths ranging from 406.7 to 514.5 nm has been used and gives rise to distinct sets of resonance Raman bands. Excitation within the Soret and Q-bands of the heme group produces the expected set of polarized and nonpolarized porphyrin modes, respectively. The frequencies of the porphyrin skeletal stretching bands in the 1450-1700 cm-1 region indicate that catalase contains only five-coordinate, high-spin heme groups. In addition to the porphyrin modes, bovine liver catalase exhibits bands near 1612 and 1520 cm-1 that are attributable to ring vibrations of the proximal tyrosinate that are enhanced via resonance with a proximal tyrosinate----Fe(III) change transfer transition centered near 490 nm. Similar bands have been observed in mutant hemoglobins that have tyrosinate axial ligands and in other Fe(III)-tyrosinate proteins. No resonance Raman bands have been observed that can be attributed to degraded hemes. The spectra are relatively insensitive to pH over the range of 5-10, and the same spectra are observed for catalase samples that do and do not contain tightly bound NADPH. Resonance Raman spectra of the fluoride complex exhibit porphyrin skeletal stretching modes that show it to be six coordinate, high spin, while the cyanide complex is six coordinate, low spin. Both the azide and thiocyanate complexes, however, are spin-state mixtures with the high-spin form predominant.     

The origin of the Adams-Schuster difference spectrum of oxyhemoglobin.

The characteristic difference spectrum reported by Adams and Schuster (Biochem. Biophys. Res. Commun. 1974, $\text{58}$, 525) on the addition of inositol hexaphosphate to oxyhemoglobin is similar to the difference spectrum between (i) isolated α- and β-chains, (ii) α- and β-semihemoglobins, (iii) addition of inorganic phosphate to oxyhemoglobin, (v) change in temperature of a solution of oxyhemoglobin, (v) change in pH of carp carboxyhemoglobin and (vi) addition of inositol hexaphosphate to α-semihemoglobin. The spectrum may also be generated by differentiation of the spectra of oxyhemoglobin and carboxyhemoglobin, implying that the common feature of the results reported above is a shift in the position of the absorption bands. This shift may arise from several causes and so its interpretation is uncertain.     

Spectral properties of myeloperoxidase and its ligand complexes

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620-630 nm is an alpha band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably 'charge-transfer' bands of the heme with the iron in the high-spin state.     

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.     

Sulfmyoglobin. Resonance Raman spectroscopic evidence for an iron-chlorin prosthetic group

The green heme protein sulfmyoglobin (SMb) has been suggested to contain a sulfur-modified iron chlorin prosthetic group. To evaluate this hypothesis, we have obtained high-frequency (greater than 1000 cm-1) resonance Raman spectra of both oxidized and reduced SMb with 457.9-, 488.0-, 514.5-, 568.2-, and 647.1-nm excitation. The SMb spectra are compared to those of native met- and deoxymyoglobin (Mb). Vibrational frequencies for SMb are generally similar to those of Mb, suggesting a high-spin state for both the Fe(III) and Fe(II) SMb species, as is typical of native Mb. However, major differences between SMb and Mb occur both for patterns of relative spectral intensities and for depolarization ratios. In particular, all B1g-depolarized porphyrin modes in the Mb spectra have become polarized, totally symmetric vibrational modes in the SMb spectra. These contrasts reflect a dramatic lowering of the effective symmetry for the SMb prosthetic group. Several new bands are observed in SMb spectra that are not present in spectra of either native Mb or iron protoporphyrin IX complexes. The observation of additional polarized bands flanking the oxidation state marker, V4, is of particular interest. In a parallel study, we compared the resonance Raman spectral properties of iron protoporphyrin IX-derived chlorins and metallo-octaethylchlorins with those of the analogous porphyrins: the chlorin spectra exhibited altered intensity patterns, an increased number of totally symmetric (polarized) vibrational bands, and several new vibrational bands, including one or two in the region of the oxidation state marker, V4. Thus, the resonance Raman spectral characteristics of SMb and metallo-chlorins are complementary and strongly support a chlorin prosthetic group for SMb. Furthermore, they establish testable criteria for investigating the prosthetic group structures of other green heme proteins by resonance Raman spectroscopy.     

Temperature dependent spectral changes of iron and nickel hemoglobins and their derivatives

Temperature dependent absolute and difference spectra for deoxy and oxy human hemoglobin, alpha and beta subunits, NiHbA, carboxypeptidase A treated deoxy HbA and NiHbA have been investigated. It is shown for the first time that the alpha subunits are mainly responsible for the temperature dependent spectral changes in the absorption spectra of Hb in the range from 0 degrees C to 40 degrees C. It has also been found that in the R state the spectral alterations caused by temperature variation are about 85% of those found for the T state of Hb. The value of following the temperature dependence of the porphyrin bands of hemoproteins, as a sensitive probe for subtle changes in the region of the heme, is demonstrated.     

Resonance Raman spectroscopy of cytochrome c peroxidase single crystals on a variable-temperature microscope stage

Good quality resonance Raman (RR) spectra have been obtained for cytochrome c peroxidase single crystals (0.2 x 0.5 x 1 mm) lying on their 110 faces on a microscope stage. Crystal orientation and polarization effects are observed which differentiate the RR bands on the basis of the symmetries of the porphyrin vibrational modes. The measured depolarization ratios are accurately calibrated for isolated bands of both totally symmetric and non totally symmetric modes by using a model of D4h chromophores in an oriented gas using the crystal structure atomic coordinates. The calculations indicate that the electronic transition moments are approximately along the lines connecting the methine bridges, suggesting an electronic steering effect of the vinyl groups. Deviations are observed for bands associated with the porphyrin v10 and the vinyl C = C stretching modes, which may be due to their near-resonant interaction. The band frequencies correspond to those of a five-coordinate high-spin FeIII heme, as previously observed in solution, consistent with the X-ray structure showing the Fe atom to be out of the heme plane on the proximal side with a distal water molecule located at a nonbonded distance, 2.4 A. The temperature dependence of the RR spectrum was determined with a Joule-Thompson cryostat on crystals sealed in glass capillaries. As the temperature is lowered, the spectrum converts to one characteristic of a low-spin FeIII heme. The conversion, which is readily reversible, is quite gradual. It is detectable at -50 degrees C but is incomplete even at -190 degrees C. A temperature effect on the protein structure is proposed which permits the Fe atom to approach the heme plane and bind the distal water molecule, or the distal histidine.     

Calculation of the energy difference between the quaternary structures of deoxy- and oxyhemoglobin.

The energy difference between the quaternary structures of deoxy- and oxyhemoglobin is evaluated on the basis of the atomic coordinates determined by X-ray diffraction analysis. Calculation of the van der Waals interaction between subunits shows that in a hemoglobin molecule as a whole, the interaction is more attractive in the oxy form than in the deoxy form by about 8 kcal/mol, and that in each pair of two subunits except the pair alpha1alpha2, the interaction energy varies by about 15 kcal/mol. The electrostatic interactions originating in the partial charges on all constituent atoms of hemoglobin and in the polar residues on the surface of hemoglobin make only a small contribution to the energy difference between the quaternary structures of deoxy- and oxyhemoglobin. Thus, the contribution of the clusters of the polar residues in the internal cavity between like subunits and also of the freedom of rotation of the C-terminal of each subunit in oxyhemoglobin may be important energetically in the transition from deoxy to oxy quaternary structure. In this point, the present calculation supports Perutz' model, but suggests necessity of further investigations on the transitional characteristics of the quaternary structure in the intermediate steps of oxygenation. The discussion on the transitional characteristics is given in the last section.     

Neuronal nitric oxide synthase ligand and protein vibrations at the substrate binding site. A study by FTIR

Improvements in sensitivity and data processing of Fourier transform infrared (FTIR) spectroscopy enable it to be used to detect changes in protein structure at the atomic level. This paper reports a study of neuronal nitric oxide synthase (nNOS) by FTIR difference spectroscopy in the 1000-2500 cm(-1) range where vibrational bands of ligands, prosthetic groups, and protein and amino acid side chains are found. We have exploited the photolyzable CO compound of the ferrous heme of nNOS to produce light-induced CO photolysis difference spectra and to compare spectra after hydrogen/deuterium exchange. In (reduced) minus (reduced plus CO) difference spectra, negative bands at 1931 and 1907 cm(-1) are observed due to photolysis of multiple forms of ferrous heme-ligated CO, similar to those observed by resonance Raman spectroscopy [Wang et al. (1997) Biochemistry 36, 4595-4606]. Photolysis of the ferrous heme CO compound is accompanied by hitherto unreported changes in the 1000-2000 cm(-1) region that arise from changes of protein backbone, substrate, amino acid side chain, and cofactor vibrations. Preliminary assignments of vibrations are made on the basis of frequencies and the effects of hydrogen/deuterium exchange, and in the light of known atomic structures.     

Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman spectra of cytochrome c oxidase. Excitation in the 600-nm region.

The resonance Raman (RR) spectra of oxidized, reduced, and oxidized cyanide-bound cytochrome c oxidase with excitation at several wavelengths in the 600-nm region are presented. No evidence is found for laser-induced photoreduction of the oxidized protein with irradiation at lambda approximately 600 nm at 195 K, in contrast to the predominance of this process upon irradiation in the Soret region at this temperature. The Raman spectra of all three protein species are very similar, and there are no Raman bands which are readily assignable to either cytochrome a or cytochrome a3 exclusively. The Raman spectra of the three protein species do, however, exhibit a number of bands not observed in the RR spectra of other hemoproteins upon exicitation in their visible absorption bands. In particular, strong Raman bands are observed in the low-frequency region of the RR spectra (less than 500 cm-1). The frequencies of these bands are similar to those of the copper-ligand vibrations observed in the RR spectra of type 1 copper proteins upon excitation in the 600-nm absorption band characteristic of these proteins. In cytochrome c oxidase, these bands do not disappear upon reduction of the protein and, therefore, cannot be attributed to copper-ligand vibrations. Thus, all the observed RR bands are associated with the two heme A moieties in the enzyme.     

Pressure effects on the proximal heme pocket in myoglobin probed by Raman and near-infrared absorption spectroscopy.

The influence of high pressure on the heme protein conformation of myoglobin in different ligation states is studied using Raman spectroscopy over the temperature range from 30 to 295 K. Photostationary experiments monitoring the oxidation state marker bands demonstrate the change of rebinding rate with pressure. While frequency changes of vibrational modes associated with rigid bonds of the porphyrin ring are <1 cm(-1), we investigate a significant shift of the iron-histidine mode to higher frequency with increasing pressure (approximately 3 cm(-1) for deltaP = 190 MPa in Mb). The observed frequency shift is interpreted structurally as a conformational change affecting the tilt angle between the heme plane and the proximal histidine and the out-of-plane iron position. Independent evidence for iron motion comes from measurements of the redshift of band III in the near-infrared with pressure. This suggests that at high pressure the proximal heme pocket and the protein are altered toward the bound state conformation, which contributes to the rate increase for CO binding. Raman spectra of Mb and photodissociated MbCO measured at low temperature and variable pressure further support changes in protein conformation and are consistent with glasslike properties of myoglobin below 160 K.     

Quantitative analysis of hemoglobin oxygenation state of rat brain in situ by near-infrared spectrophotometry

The light in the near-infrared region (700-900 nm) was illuminated on the rat head, and absorption spectra were measured with the transmitted light under various conditions. The absorbance changes less than 780 nm were attributable to hemoglobin in the brain tissue, whereas those greater than 780 nm were associated with both hemoglobin and cytochrome oxidase. The changes of oxy- and total (oxy- plus deoxy-) hemoglobin content in the rat head could be monitored quantitatively by expressions of delta A700--1.20 delta A730 and delta A700--1.52 delta A730, respectively. The oxyhemoglobin content in the tissue was decreased as the O2 tension in inspired gas was lowered. At 10% O2 approximately 50% of hemoglobin was deoxygenated. The total hemoglobin content was increased under anoxic conditions. Inhalation of 5% CO2 and intravenous injection of a Ca2+ blocker nicardipine increased the O2 saturation of hemoglobin in the brain. These conclusions were confirmed by measuring the difference absorption spectra in the near-infrared region.     

Partial reduction of aquomethemoglobin on a sephadex G-25 column as detected by magnetic circular dichroism spectroscopy and revised extinction coefficients for aquomethemoglobin

In a typical preparation of aquomethemoglobin, oxyhemoglobin is oxidized with potassium ferricyanide, and the resultant mixture of methemoglobin and potassium ferro- and ferricyanides is separated on a Sephadex G-25 column. We find that about 1% of the heme is reduced on the column and is eluted with the methemoglobin. Magnetic circular dichroism spectra show that the reduced species is oxyhemoglobin. Magnetic circular dichroism is more sensitive than is absorption spectroscopy to small amounts of oxyhemoglobin in such solutions; we can detect its presence at the 0.1% level. A redetermination of the extinction coefficients for methemoglobin gives a value of 0.80 for the absorbance ratio A⁵⁷⁰/A⁶³⁰ at pH 6. This value lies clearly outside the currently accepted range of 0.83 to 0.87.     

Magnetic circular dichroism studies of bovine liver catalase.

Absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of beef liver catalase at pH 5.0 and 6.9, and its complexes with NaF, KCNO, NaCNS, NaN3 and NaCN, have been measured between 250 nm and 700 nm at room temperature. The pH 6.9 native catalase MCD shows the presence of several additional transitions not resolved in the absorption spectrum. While these bands can be seen in the spectra of all the derivatives, with the exception of the cyanide, their relative intensities changes considerably between complexes. Of special interest in the MCD of ferric hemes is the signal intensity at about 400 nm and 620 nm. The data indicate that the MCD intensity at 620 nm increases as the high spin iron porphyrin fraction increases, reaching a maximum with the fluoride complex. The 430 nm band intensity increases as the proportion of low spin iron increases, reaching a maximum with the cyanide complex. The MCD spectra also indicate clearly the existence of spin mixtures in the complexes with CNO-, CNS-, and N3-, where both the 430 nm and 620 nm bands have appreciable intensity. It is significant that despite almost identical absorption spectra the CNS- complex has higher fraction of low spin iron than either the CNO- or the N3- species. The differences between the pH 5 and 6.9 MCD spectra of the native catalase suggest that the environment of the heme centre is sensitive to protonation.