Spin equilibria in human methemoglobin: effects of bezafibrate and inositol hexaphosphate as measured by susceptometry and visible spectroscopy

The effects of inositol hexaphosphate (IHP) and a second allosteric effector, bezafibrate, on the spin-state equilibria of the mixed-spin derivatives of ferric human hemoglobin A are examined. Changes in spin-state equilibrium are monitored by measuring absorption spectra in the visible region (460-700 nm) as well as by direct measurements of magnetic susceptibility by means of a superconducting fluxmeter. The addition of IHP at pH 6.5 results in a measurable shift in the spin equilibria of these derivatives toward higher spin. However, the addition of bezafibrate in the presence of IHP results in still larger shifts toward the high-spin form. The changes in the free energies of the spin-state equilibria resulting from the combination of these two effectors are similar in magnitude to that which results from the R-state to T-state transition in carp hemoglobin.     

Binding of inositol hexaphosphate to human methemoglobin.

The observed static difference spectrum produced by inositol hexaphosphate binding to methemoglobin is the sum of a very fast and a slow spectral transition. The more rapid absorbance change is too fast to be measured by stopped flow techniques, whereas the slow change exhibits a half-time in the range 1 to 6 s. From the pH dependence of the rapidly formed difference spectrum and from a series of heme ligand binding studies, the rapid phase is interpreted to reflect a localized tertiary conformational change which immediately accompanies inositol hexaphosphate binding and results in a selective increase in spin and reactivity of the beta chain heme groups. In contrast, the slow phase appears to reflect a first order isomerization process which involves only a small portion (less than 10%) of the hemoglobin molecules and results primarily in a marked alteration of the spectral properties of the alpha chains with little change in spin. While the rapid spectral transition cannot be directly related to the overall quaternary transition which occurs during oxygen binding to ferrous deoxyhemoglobin, the slow spectral transition may represent the abortive formation of a deoxyhemoglobin A-like conformation which is inhibited in both rate and extent by the presence of water molecules bound to the heme iron atoms.     

Quaternary equilibrium analysis of the imidazole methemoglobin bound with inositol hexaphosphate

The visible and proton NMR spectral responses of imidazole methemoglobin by the binding of inositol hexaphosphate were examined in the 2-40 degrees C range. The magnitude of the +/- (inositol hexaphosphate) visible difference spectrum increased and the intensity of the 33 ppm NMR peak decreased with lowering of the temperature. The NMR results were quantitatively analyzed with a simple two-state allosteric model. The results show that the T conformer fraction is 0.6 at 20 degrees C and that the equilibrium shifts toward the T state at lower temperature. The large changes in delta H and delta S associated with the equilibrium suggest participation of numerous factors in the determination of the equilibrium position. The increase in the T conformer population of imidazole methemoglobin, which is pure low-spin, suggests that the appearance of the T state with decreasing temperature is not directly coupled to an increase in spin of the heme iron.     

Interaction of human adult methemoglobin in low-spin state with inositol hexaphosphate. A proton magnetic resonance study

The hyperfine-shifted proton nuclear magnetic resonance (NMR) spectra of the low-spin complexes of human adult methemoglobin were found to be much altered by the addition of inositol hexaphosphate (IHP). The stoichiometry and pH-dependence of IHP binding, and the spin equilibrium of azide methemoglobin are parallel to those of high-spin human methemoglobin and of carp methemoglobin, both of which are proposed to be switched from the R to T states with IHP. The present NMR results show that IHP affects the structure of human methemoglobin regardless of the spin state of the heme iron, suggesting that there is no correspondence between quaternary structure and the spin state of ferric heme iron.     

Influence of inositol hexaphosphate binding on subunit dissociation in methemoglobin.

The tetramer-dimer equilibria of various forms of methemoglobin have been measured by sedimentation equilibrium to test the hypothesis of Perutz that high spin derivatives can be switched by inositol hexaphosphate (Inos-P6) from the R state to the T state more readily than low spin derivatives. Since transitions from the R state to the T state are accompanied by a decrease in the tetramer-dimer dissociation constant (K4,2), this parameter is a quantitative indicator of the conformational state. Measurements of K4,2 were performed using an analytical ultracentrifuge with absorption optics and a scanner-computer system. Statistical analysis of the sedimentation data indicated that the stoichiometry if Inos-P6 binding is 1 molecule/hemoglobin tetramer and 2 molecules/hemoglobin dimer. The apparent affinity of the dimer sites for Inos-P6 is much lower than the corresponding value for the tetramer site. As a result of the stoichiometries, at low concentrations Inos-P6 shifts the tetramer-dimer equilibrium in favor of the tetramer, but at high concentrations Inos-P6 shifts the equilibrium in favor of the dimer. Te tetramer binding site for Inos-P6 of various liganded forms of hemoglobin appears to be the same as has been established for deoxyhemoglobin, since the effect of Inos-P6 on subunit dissociation is reduced in pyridoxylated derivatives. Values of K4,2 for aquo-, azido- and cyanomethemoglobin in 0.01 M 2,2-bis(hydroxymethyl)-2,2',2'-nitroethanol buffer, pH 6.0/0.1 M NaCl, are all near 2 X 10(-5) M. Upon addition of 50 muM Inos-P6 the values of K4,2 for all three forms are shifted to near 10(-9) M. Since the aquo derivative is high spin, while the azido and cyano derivatives are low spin, the similarity of values for the derivatives in the presence and absence of Inos-P6 indicate that the changes in K4,2 are not spin-spin state dependent. For another high spin derivative, fluoromethemoglobin, such high concentrations of NaF are required that ionic strength effects are encountered. When data at several NaF concentrations are extrapolated to 0.1 M NaF to correct for the ionic strength effects, values of K4,2 of 7 X 10(-6) M and 10(-8) M are obtained for solutions in the absence and in the presence of 50 muM Inos-P6, respectively. Therefore the results with the fluoro derivative, in conjunction with the other forms of methemoglobin, support the view that high spin derivatives do not exhibit a greater response to Inos-P6 than low spin derivatives.     

Nuclear relaxation studies on human methemoglobin. Observation of cooperativity and alkaline Bohr effect with inositol hexaphosphate.

Ehanced spin-lattice relaxation (1/t1) of water protons induced by the heme iron of human aquomethemoglobin is exchanged-limited (koff = 1.4 times 10-4 per s at 30 degrees, H+ =7.5 Cal per mol) as indicated by the temperature and frequencey dependencies. A comparison of deuteron and proton relaxation rates revealed an order of magnitude primary isotope effect and a small inverse secondary isotope effect on the escape rate of protons from the heme iron into bulk water establishing the exchange of protons and not the exchange of the entire water molecule to be the chemical mechanism of the entire water molecule to be the chemical mechanism of the exchange process. With fluoromethemoglobin, the relaxation rate is in the fast exchange region. The results can be understood in terms of a water molecule interacting with the heme iron at an iron to proton distance less than 3.4 A in aquomethemoglobin and a single proton at a distance of 4.11 A assignable to the NH proton of the distal histidine imidazole group in fluoromethemoglobin. The relaxation rates are pH-dependent and normal titrations with Hill coefficients n = 1 are observed. The pKa is less than or equal to 6. 7 with aquomethemoglobin and 8.5 with fluoromethemoglobin at 30 degrees C. The binding of inositol hexaphosphate in stoichiometric amounts has no significant effect on the magnetic susceptibility of solutions of aquomethemoglobin and fluoromethemoglobin, but in the former case it increases koff to 3.8 times 10-4 per s by lowering the H+ barrier to 6.8 Cal per mol. In fluoromethemoglobin, inositol hexaphosphate decreases the iron to distal histidine NH distance by 0.17 A and the electron relaxation time taus by 10% as determined by the frequency dependence of 1/T1. In the aquomethemoglobin system, inositol hexaphosphate induces a Bohr effect, raising the pKa of the ionization responsible for the 1/T1 titration to 7.2, and induces cooperativity in the pH titration with a Hill coeffocoemt n = 2.8 plus or minus 0.1. With fluoromethemoglobin, the normal pH titration curve is unaffected by inositol hexaphosphate (n approximately equal to 1). Further, relaxivity titrations with varying amounts of azide and fluoride near neutral pH show normal behavior (n = 1) with and without inositol hexaphosphate. These results indicated that inositol hexaphosphate alters the quaternary structure of methemoglobin to the deoxy conformation without causing a change in the spin state of the heme iron...     

Nitrosylhemoglobin Wood: effects of inositol hexaphosphate on thiol reactivity and electron paramagnetic resonance spectrum.

Properties of Hb Wood (beta-97(FG4)His leads to Leu), a high oxygen affinity hemoglobin with reduced hemeheme interaction, were examined in its nitric oxide liganded form. The reactivity of the beta-93 thiol groups and the electron paramagnetic resonance (EPR) spectrum were examined to determine what effect the amino acid substitution, which occurs at the alpha1beta2 interface, would have on inositol hexaphosphate induced transition of this form of the tetramer. Binding of inositol hexaphosphate (IHP) in a 1:1 stoichiometry was demonstrated. In spite of apparently normal interaction with IHP, there was little or no change in the reactivity of the beta-93 thiol groups and in the electron paramagnetic resonance (EPR) spectrum as contrasted with the marked changes characteristic of normal hemoglobin (HbA). In contrast with NO-HbA, there was also no development of the EPR hyperfine structure in NO-Hb Wood with increased protonation of the protein at pH below 7.0. Taken together with the observations of Henry and Banerjee ((1973), J. Mol. Biol. 73, 469) on the development of NO-Hb EPR hyperfine structure and of Perutz et al. (1974a), Biochemistry 13, 2174) on changes in thiol reactivity with the R leads to T transition, the results suggest that IHP or H+ cannot switch NO-Hb Wood to the T conformation. Since the atomic structures of met- and deoxyhemoglobin offer no indication that His-97 plays any special part in the allosteric mechanism (M. E. Perutz, personal communication), it appears that the replacement of His-97 by Leu reduces the stability of the T structure relative to that of R.     

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subpicosecond dynamics in nucleotides measured by spontaneous Raman spectroscopy

The band widths in Raman spectra are sensitive to dynamics active on a time scale from 0.1 to 10 ps. The band widths of nucleotide vibrations and their dependence on temperature, concentration, and structure are reported. From the experimental band widths and second moments, it is derived that the adenine vibrations at 725, 1336, 1480, and 1575 cm⁻¹, and the uracil vibration at 787 cm⁻¹, are in the fast modulation limit. The correlation times of the perturbations are faster than 0.4 ps. Thermal melting of the helical structure in polynucleotides results in larger band widths, due to an increase in vibrational dephasing and energy relaxation as a consequence of the increased interaction of the base moieties with the solvent molecules. The band width of the 725 cm⁻¹ adenine vibration is dependent on the type and structure of the backbone. It is found to be perturbed by movements of the sugar-phosphate moiety relative to the base. The band width of the 1575 cm⁻¹ adenine vibration is found to be sensitive to the base-pairing interaction. From a comparison of the band widths in polynucleotides with a different base sequence (homopolymer vs alternating purine-pyrimidine sequence), it is concluded that resonant vibrational energy transfer between the base molecules is not important as a relaxation process for the vibrational band widths of nucleotides. Several theoretical models for the interpretation of band widths are discussed. The theory does not take into account the strong hydrogen-bonding nature water and hence fails to describe the observations in nucleotide-water systems. The bands of the carbonyl stretching vibrations are inhomogeneously broadened. The carbonyl groups have a strong dipolar interaction with the polar water molecules and are therefore strongly perturbed by coupling to the heatbath via hydrogen bonds. © 1997 John Wiley & Sons, Inc. Biopoly 41: 751–763, 1997     

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.     

Amide proton exchange in human metallothionein-2 measured by nuclear magnetic resonance spectroscopy

In human metallothionein-2, the exchange rate constants of ten amide protons were found to range from 1.7 x 10(-4) to 1 x 10(-1) min-1 at pH 6.3 and 8 degrees C. Most of these slowly exchanging protons could be associated with hydrogen bonds in secondary structure elements of the alpha-domain. Amide proton exchange rates thus present an additional criterion for the structural characterization of different metallothioneins, which could be particularly valuable for comparisons of different homologous protein preparations containing nuclear magnetic resonance-inactive metal ions, where the metal-polypeptide co-ordinative bonds cannot be identified directly.     

Elucidation of inositol hexaphosphate and heparin interaction sites and conformational changes in arrestin-1 by solution nuclear magnetic resonance

Arrestins specifically bind activated and phosphorylated G protein-coupled receptors and orchestrate both receptor trafficking and channel signaling through G protein-independent pathways via direct interactions with numerous nonreceptor partners. Here we report the first successful use of solution NMR in mapping the binding sites in arrestin-1 (visual arrestin) for two polyanionic compounds that mimic phosphorylated light-activated rhodopsin: inositol hexaphosphate (IP6) and heparin. This yielded an identification of residues involved in the binding with these ligands that was more complete than what has previously been feasible. IP6 and heparin appear to bind to the same site on arrestin-1, centered on a positively charged region in the N-domain. We present the first direct evidence that both IP6 and heparin induced a complete release of the arrestin C-tail. These observations provide novel insight into the nature of the transition of arrestin from the basal to active state and demonstrate the potential of NMR-based methods in the study of protein-protein interactions involving members of the arrestin family.     

Interaction of adriamycin with DNA as studied by resonance Raman spectroscopy.

Raman and resonance Raman spectra of the complex DNA-adriamycin in aqueous solution have been recorded and analysed. Calf thymus DNA was used and it is found that in the complex DNA-adriamycin the chromophore of adriamycin is intercalated in the GC sequences. The substituents on the rings give hydrogen bonding interactions with the base pairs above and below the intercalation site. It is suggested from the Raman and resonance Raman spectral modifications that the phenolic groups of the chromophore are involved in the drug-DNA intercalation, in addition to pi-pi, hydroxyl and amino group interactions.     

Interaction of fully liganded valency hybrid hemoglobin with inositol hexaphosphate. Implication of the IHP-induced T state of human adult methemoglobin in the low-spin state

To gain further insight into the quaternary structures of methemoglobin derivatives in the low-spin state, the interaction of fully liganded valency hybrid human hemoglobins with IHP was studied by proton NMR spectroscopy. Upon addition of IHP to (alpha CO beta + N3-)2, the same resonances as the previously reported IHP-induced NMR peaks for azidomethemoglobin (alpha + N3-beta +N3-)2 appeared, whereas the binding of IHP did not significantly affect the NMR spectra for (alpha + N3-beta CO)2. The binding of IHP also brought about more pronounced spectral changes for (alpha CO beta + Im)2 and (alpha CO beta + H2O)2 than for (alpha + Im beta CO)2 and (alpha + H2O beta CO)2. Therefore, the IHP-induced NMR peaks for azidomethemoglobin are attributed to the beta heme methyl group. Such IHP-induced beta heme methyl resonances were also observed for (alpha NO beta + N3-)2, which undergoes quaternary structural change, analogously to the R-T transition by the binding of IHP. From the above results, it was suggested that the IHP-induced heme methyl resonances for azidomethemoglobin and (alpha CO beta +N3-)2 may also be associated with the quaternary structure of these Hbs, implying the presence of the IHP-induced "T-like" state in low-spin metHb A.     

Effect of inositol hexaphosphate on hemoglobin oxidation by nitrite and ferricyanide

In the presence of inositol hexaphosphate (IHP), the rate of hemoglobin oxidation by nitrite was much inhibited; however, that of the hemoglobin oxidation by ferricyanide was much accelerated. The difference in the reaction mode was discussed in relation to the interaction of hemoglobin with IHP. The dissociation constant of IHP to oxyhemoglobin was estimated from the rate of the hemoglobin oxidation by ferricyanide in different concentrations of IHP under oxygen saturated conditions.     

Interactions of soluble guanylate cyclase with diatomics as probed by resonance Raman spectroscopy

Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm(-1), but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (nuFe-CO) mode at 473 and 489 cm(-1), both of which are His- and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (nuFe-CO=489 cm(-1)) in addition to 5c CO-adduct (nuFe-CO=521 cm(-1)). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of gamma-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.