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.     

Glass transition of deoxymyoglobin probed by optical absorption spectroscopy

We have measured the absorption spectrum of horse deoxymyoglobin in glycerol-water mixture around 430 nm in the 130 - 320 K temperature range. The observed asymmetric spectral shape of the Soret band was analyzed using a configuration-coordinate model. The results support the idea that myoglobin is liquid-like at physiological temperatures, but is glass-like below about 250 K. The equilibrium position of the iron atom in the heme group in the electronic excited state was estimated from the determined parameter values.     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.     

Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH

Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the ‘rhombic’ fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the ‘axial’ form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.     

Rebinding and relaxation in the myoglobin pocket

The infrared stretching bands of carboxymyoglobin (MbCO) and the rebinding of CO to Mb after photodissociation have been studied in the temperature range 10-300 K in a variety of solvents. Four stretching bands imply that MbCO can exist in four substates, A0-A3. The temperature dependences of the intensities of the four bands yield the relative binding enthalpies and and entropies. The integrated absorbances and pH dependences of the bands permit identification of the substates with the conformations observed in the X-ray data (Kuriyan et al., J. Mol. Biol. 192 (1986) 133). At low pH, A0 is hydrogen-bonded to His E7. The substates A0-A3 interconvert above about 180 K in a 75% glycerol/water solvent and above 270 K in buffered water. No major interconversion is seen at any temperature if MbCO is embedded in a solid polyvinyl alcohol matrix. The dependence of the transition on solvent characteristics is explained as a slaved glass transition. After photodissociation at low temperature the CO is in the heme pocket B. The resulting CO stretching bands which are identified as B substates are blue-shifted from those of the A substates. At 40 K, rebinding after flash photolysis has been studied in the Soret, the near-infrared, and the integrated A and B substates. All data lie on the same rebinding curve and demonstrate that rebinding is nonexponential in time from at least 100 ns to 100 ks. No evidence for discrete exponentials is found. Flash photolysis with monitoring in the infrared region shows four different pathways within the pocket B to the bound substates Ai. Rebinding in each of the four pathways B----A is nonexponential in time to at least 10 ks and the four pathways have different kinetics below 180 K. From the time and temperature dependence of the rebinding, activation enthalpy distributions g(HBA) and preexponentials ABA are extracted. No pumping from one A substate to another, or one B substate to another, is observed below the transition temperature of about 180 K. If MbCO is exposed to intense white light for 10-10(3) s before being fully photolyzed by a laser flash, the amplitude of the long-lived states increases. The effect is explained in terms of a hierarchy of substates and substate symmetry breaking. The characteristics of the CO stretching bands and of the rebinding processes in the heme pocket depend strongly on the external parameters of solvent, pH and pressure. This sensitivity suggests possible control mechanisms for protein reactions.     

Interaction of aromatic donor molecules with lactoperoxidase probed by optical difference spectra

On the basis of optical difference spectra, lactoperoxidase (LPO) was shown to form a 1:1 complex with aromatic donor molecules: resorcinol, hydroquinone, phenol, p-cresol, guaiacol, aniline, and benzohydroxamic acid. As compared with horseradish peroxidase (HRP), the values of the dissociation constant, Kd, of LPO-donor complexes were found to be 4-720-fold larger and were not greatly changed in the presence of KCN and by changes in pH in the range 4-9. The apparent enthalpy and entropy of the binding reactions were found to be -13 kJ mol-1 and -29 J mol-1 K-1, respectively, somewhat smaller (in absolute value) than the corresponding values of HRP. The difference spectra of LPO-donor complexes resembled each other, in contrast to the case of HRP, and the bindings of the donors to LPO occurred in a competitive fashion between the donors. Incubation of LPO with phenylhydrazine and hydrogen peroxide markedly depressed donor binding, the intensity of the Soret band, and the catalytic activity, probably as the result of formation of meso-phenyl derivatives of the heme. These findings suggest that the binding of aromatic donors to LPO occurs at a specific site, probably near the heme edge, where the electron transfer in the peroxidase reaction may take place.     

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.     

Variations of electrostatic interactions in myoglobin probed by UVRR spectroscopy. Effect of iron ligand and pH on tryptophan and tyrosine vibrations.

The present work investigates the variations of electrostatic interactions within the myoglobin molecule associated with azide heme binding and pH variations. Far ultraviolet (223 nm) resonance Raman spectroscopy of the tryptophan and tyrosine residues, along with acid-base titration measurements, have been used to monitor variations in the protein matrix. With previously determined mode assignments, it is shown that the Trp and Tyr residues of the globin moiety are influenced by the charge spatial distribution. Upon ligand binding or under various pH conditions, the polar interactions inside the protein appear to be modulated by the electric field generated by the charge array. It is concluded that the binding site properties of myoglobin can be modulated by the charge spatial distribution within the protein, even in the absence of measurable conformational changes of the bulk.     

Protein and chlorophyll in photosystem II probed by infrared spectroscopy

The infrared spectra of photosystem II (PS II) enriched submembrane fractions isolated from spinach are obtained in water and in heavy water suspension Other spectra are obtained after a photooxidation reaction was performed on PS II to bleach the pigments. The water bands are removed by computer subtraction and the amide bands (A, B, I, II, and III) of the protein are identified. Computer enhancement techniques are used to narrow the bandwidth of the bands that the weak chlorophyll bands, buried in the much stronger protein bands, can be observed. Comparing the spectra of native and photooxidized PS II pr in water and in heavy water, we determine that three polypeptide domains are present in the native material. The first domain, which contains 22% of th is situated in the peripheral region of the PS II system. The polypeptides in this region are unfolded and devoid of chlorophyll. The second domain con of the polypeptides, is more organized, and contains the chlorophylls. The third domain has an alpha-helix configuration, does not contain chlorophyll, a affected by the photooxidation reaction or by the proton/deuteron exchange. Three different types of chlorophyll organisation are identified: two have carbonyl groups non-bonded, differing from one another only in their hydrophobic milieux; the third is weakly bonded to another unidentified group. Other forms of chlorophyll organisation are present but could not be observed because their absorption is buried in the protein amide I band.     

Interaction process between ionic surfactant and protein probed by series piezoelectric quartz crystal technique

A method for probing the interaction process between ionic surfactant and protein was developed with series piezoelectric quartz crystal (SPQC) sensing technique. It was based on the sensitive response of the SPQC sensor to the change in solution conductivity. A new relationship between the sensor response and the properties of ionic species in solution was derived. The method was used to examine the interaction process of two surfactants, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), with lysozyme in aqueous solution. The obtained experimental results were in agreement with those of other methods from references. These results had been discussed. It was shown that the new method developed here was a useful and promising tool for probing the ionic surfactant-protein interaction process and might find more applications in similar studies.     

The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy.

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.     

Local magnetism in palladium bionanomaterials probed by muon spectroscopy

Palladium bionanomaterial was manufactured using the sulfate-reducing bacterium, Desulfovibrio desulfuricansm, to reduce soluble Pd(II) ions to cell-bound Pd(0) in the presence of hydrogen. The biomaterial was examined using a Superconducting Quantum Interference Device (SQUID) to measure bulk magnetisation and by Muon Spin Rotation Spectroscopy (μSR) which is uniquely able to probe the local magnetic environment inside the sample. Results showed behaviour attributable to interaction of muons both with palladium electrons and the nuclei of hydrogen trapped in the particles during manufacture. Electronic magnetism, also suggested by SQUID, is not characteristic of bulk palladium and is consistent with the presence of nanoparticles previously seen in electron micrographs. We show the first use of μSR as a tool to probe the internal magnetic environment of a biologically-derived nanocatalyst material.     

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.     

Conformational differences between various myoglobin ligated states as monitored by 1H NMR spectroscopy

In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-dependent forms of the ferryl haem in myoglobin peroxide analysed by variable-temperature magnetic circular dichroism.

The reaction product of myoglobin and H2O2 exists in two different forms according to the external pH. Varied-temperature magnetic-circular dichroism (m.c.d.) spectroscopy demonstrates that both contain the oxyferryl ion Fe(IV) = O. Alkaline myoglobin peroxide has often been used as a model for oxidized intermediates in the catalytic cycles of haem-containing peroxidases, but absorption and m.c.d. spectra show that the acid form is much more closely related to species such as horeradish peroxidase Compound II. The differences are tentatively ascribed to ionization of the proximal histidine ligand in alkaline myoglobin peroxide. It is also shown that the m.c.d. method allows an estimate of the zero-field splitting parameter of both forms, values of D = 28.0 +/- 3 cm-1 and 35.0 +/- 5 cm-1 being obtained for the alkaline and acid forms respectively.     

Heme structural perturbation of PEG-modified horseradish peroxidase C in aromatic organic solvents probed by optical absorption and resonance Raman dispersion spectroscopy

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B(1g) distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B(2g)-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP.     

Blue-light-induced changes in Arabidopsis cryptochrome 1 probed by FTIR difference spectroscopy

Cryptochromes are blue-light photoreceptors that regulate a variety of responses in animals and plants, including circadian entrainment in Drosophila and photomorphogenesis in Arabidopsis. They comprise a photolyase homology region (PHR) of about 500 amino acids and a C-terminal extension of varying length. In the PHR domain, flavin adenine dinucleotide (FAD) is noncovalently bound. The presence of a second chromophore, such as methenyltetrahydrofolate, in animal and plant cryptochromes is still under debate. Arabidopsis cryptochrome 1 (CRY1) has been intensively studied with regard to function and interaction of the protein in vivo and in vitro. However, little is known about the pathway from light absorption to signal transduction on the molecular level. We investigated the full-length CRY1 protein by Fourier transform infrared (FTIR) and UV/vis difference spectroscopy. Starting from the fully oxidized state of the chromophore FAD, a neutral flavoprotein radical is formed upon illumination in the absence of any exogenous electron donor. A preliminary assignment of the chromophore bands is presented. The FTIR difference spectrum reveals only moderate changes in secondary structure of the apoprotein in response to the photoreduction of the chromophore. Deprotonation of an aspartic or glutamic acid, probably D396, accompanies radical formation, as is deduced from the negative band at 1734 cm(-)(1) in D(2)O. The main positive band at 1524 cm(-)(1) in the FTIR spectrum shows a strong shift to lower frequencies as compared to other flavoproteins. Together with the unusual blue-shift of the absorption in the visible range to 595 nm, this clearly distinguishes the radical form of CRY1 from those of structurally highly homologous DNA photolyases. As a consequence, the direct comparison of cryptochrome to photolyase in terms of photoreactivity and mechanism has to be made with caution.     

Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.