The thermodynamics of myoglobin stability. Effects of axial ligand.

The reversible thermal denaturation of four metmyoglobin derivatives, aquomet, cyanomet, azidomet and fluoromet in both the alkaline pH and acidic pH region, has been examined by optical spectrophotometry. The data are analyzed in terms of a two-state model to extract the thermodynamic parameters delta G, delta H, delta S and delta Cp characterizing the transition. The results are consistent with Brandts' phenomenological model of protein denaturation. Among the derivatives examined, cyanomet, azidomet and fluoromet are about 3 kcal/mol, 2 kcal/mol and 0.2 kcal/mol, respectively, more stable than aquometmyoglobin. The observed differences are found to be inconsistent with the hypothesis that the stability is mainly governed by the spin state of the iron atom. In addition, the enthalpic and entropic contributions in delta G are extracted and the differences in delta G for the various derivatives are found to arise from minor changes in delta H and T delta S. Assuming the final denaturated state to be universal, these changes reflect the effect of ligands on the conformational energy of the native protein.

Parameters controlling the kinetics of ferric and ferrous hemeproteins reduction by hydrated electrons.

To clarify the processes of hemeproteins reduction, three classes of these proteins (ferric, ferrous and desFe) were reduced by hydrated electrons generated by pulse radiolysis. Spectral and kinetic investigations were made on alpha hemoglobin chain and myoglobin. Human alpha chain has been chosen to avoid all ferric contaminations and horse ferric myoglobin to eliminate all ferrous protein fractions. We have successively studied the influences of: the iron presence, its oxidation state (II and III), the protein charge and the iron-ligand nature (H2O, OH-, N3- and CN-). For alpha human hemoglobin chain without metallic ion or with ferrous iron, the reduction rates are the same: 1.1 +/- 0.2.10(10) M-1.s-1. In the case of horse ferric myoglobin, the reduction rates depend principally on the protein charge (from pH 6.3 to pH 9.5, the reduction rate of Mb(FeIII)N3- decreases from 2.5 +/- 0.5.10(10) M-1.s-1 to 1.2 +/- 0.2.10(10) M-1.s-1) and are also modulated by the equilibrium constant of the hemeprotein-ligand association (1.2 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)N3- and 0.8 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)CN-, at pH 9.8).

Resonance Raman spectroscopic studies of ligated mutant myoglobins.

Ligand binding (CO and N3-) to wild-type porcine myoglobin and to several mutant forms, expressed and purified from E. coli cells, has been studied using Raman spectroscopy. The v(Fe-CO) stretching vibration in MbIICO has been compared for the wild-type and mutant proteins. This gives a broad band consisting of five components, indicating five possible configurations of the bound CO. The distal pocket mutants show large variations in bandshape, the major component occurring at progressively lower wavenumber in the order: wild-type (WT) > E11 Val-->Thr (VT) > E7 His-->Val (HV) > the double mutant VT/HV (M2). Changes observed in the Raman band assigned to the azide bending mode in MbIIIN3 have been interpreted in terms of resonance structures involving two forms of azide binding. Repulsion between the bound azide ligand and the OH group of the adjacent thr residue in the VT mutant, and a shorter Fe-N(his) bond in the proximal mutant Ser-->Leu (F7), both affect this bonding. In the wild-type protein (WT), hydroxymetmyoglobin exists in a spin-state equilibrium which, at room temperature, is predominantly high-spin. In the F7 mutant this equilibrium is shifted in favour of the low-spin form. A low-spin iron species also exists in the aquometmyoglobin form of this mutant.

Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.

The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.

Transition state of an unfolding step in human cyanomet myoglobin.

We studied an unfolding step of cyanomet myoglobin (Mb) unfolding, for demonstrating dynamical structural changes in the transition state of the process. Three leucine-->alanine mutant Mbs (L29A, L72A and L104A) were prepared for this study. The urea-induced largely monophasic process was monitored by absorption spectroscopy. Linear relations between [urea] and the activation energy (delta G not equal to) of the relaxation for all the Mbs showed that the slope m not equal to urea (= delta(delta G not equal to)/delta[urea])) was altered by either reduction of pH or the L-->A mutations. Thermodynamic interpretations of the changes in m not equal to urea led to a conclusion that the exposed surface area of Mb in the transition state was determined by both protein-core stability and pH conditions. We also performed urea- and acid-denaturation experiments, and gave some inspections on differences between mutational effects on the structure of the transition state and the denatured state.

NMR study of the molecular and electronic structure of the heme cavity in Dolabella met-cyano myoglobin.

The molecular and electronic structure of the active site of the cyanide-ligated ferric complex of the myoglobin from the mollusc Dolabella auricularia has been investigated using NMR. Analysis of nuclear Overhauser effects has revealed that the correlation times for the internal motion of the heme propionate alpha-CH2 and beta-CH2 groups at ambient temperature are about 5 and 4 ns, respectively. These correlation times indicate that the terminal carboxylate groups of both the heme propionates are not bound to the protein via salt bridges. Although the absence of the propionate-protein interaction does not influence the equilibrium population of the two heme orientational isomers involving rotation about the alpha,gamma-meso axis, it allows the heme to rotate about the iron-His bond in the active site of the myoglobin. Such rotational motion of the heme resulted in an anomalous temperature-dependence of the heme methyl-proton hyperfine shift. Thus the present myoglobin studies provide the first example demonstrating the rotation of the heme about the iron-His bond in native myoglobin.

Hydrogen bonding interaction of the amide group of Asn and Gln at distal E7 of bovine myoglobin with bound-ligand and its functional consequences.

Asn and Gln with an amide group at gamma- and delta-positions, respectively, were substituted for distal His-E7 of bovine myoglobin to establish a system where hydrogen bonding interaction between the distal residue and bound-ligand can be altered by changing donor-acceptor distance. Two mutant myoglobins showed nearly identical (1)H-NMR spectral pattern for resolved heme peripheral side-chain and amino acid proton signals and similar two-dimensional NMR connectivities irrespective of cyanide-bound and -unbound states, indicating that the heme electronic structure and the molecular structure of the active site are not affected by a difference in one methylene group at the E7 position. Chemical exchange rate of Asn-E7 N(delta)H proton in met-cyano myoglobin is larger than that of Gln-E7 N(epsilon)H proton by at least two orders of magnitude, suggesting a considerable difference in the strength of hydrogen bond between the E7 side-chain and bound-ligand, due to the differential donor-acceptor distance between the two mutants. Thus a comparative study between the two proteins provides an ideal system to delineate a relationship between the stabilization of bound-ligand by the hydrogen bond and myoglobin's ligand affinity. The Asn-mutant showed a faster dissociation of cyano ion from met-myoglobin than the Gln-mutant by over 30-fold. Similarly, oxygen dissociation is faster in the Asn-mutant than in the Gln-mutant by approximately 100-fold. Association of cyanide anion to the mutant met-myoglobin was accelerated by changing Gln to Asn by a 4-fold. Likewise, oxygen binding was accelerated by approximately 2-fold by the above substitution. The present findings confirm that hydrogen bonding with the distal residue is a dominant factor for determining the ligand dissociation rate, whereas steric hindrance exerted by the distal residue is a primary determinant for the ligand association.

Structural and electronic properties of the liver fluke hemoglobin heme cavity by nuclear magnetic resonance: hemin isotope labeling.

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.

Heme orientational disorder in reconstituted and native sperm whale myoglobin. Proton nuclear magnetic resonance characterizations by heme methyl deuterium labeling in the Met-cyano protein.

The solution proton nuclear magnetic resonance spectrum of the Met-cyano form of sperm whale myoglobin reveals the presence of two sets of comparably intense resonances immediately after reacting the apoprotein with hemin, only one of which corresponds to that of the accepted native protein. Isotope labeling of individual methyl groups of hemin reveals that the methyl assignments differ characteristically in that similar resonance positions for the two components arise from the methyl groups related by a 180 degrees rotation about the alpha-gamma-meso axis. This phenomenon, observed earlier only for myoglobin with modified hemin, dictates that the second protein component in solution immediately after reconstitution must have the heme rotated by 180 degrees about the alpha-gamma-meso axis as compared to that found in the single crystal. The two components in the reconstituted protein equilibrate to yield the spectrum of the native Met-cyanomyoglobin for which there still exists approximately 8% of the minor component. Thus native myoglobin in solution is structurally heterogeneous in the heme pocket. Proton nuclear magnetic resonance spectra of deoxymyoglobin produced from both native and freshly reconstituted protein shown that the heterogeneity is also a property of the physiologically relevant reduced protein forms. It is suggested that, contrary to available X-ray data, heme orientational heterogeneity may be the rule rather than the exception in b-type hemoproteins, and that such disorder must be carefully considered in detailed correlations between structure and function even in native hemoproteins.

Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution.

The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.

Assignment of hyperfine shifted haem methyl carbon resonances in paramagnetic low-spin met-cyano complex of sperm whale myoglobin.

The hyperfine shifted resonances arising from all four individual haem carbons of the paramagnetic low-spin met-cyano complex of sperm whale myoglobin have been clearly identified and assigned for the first time with the aid of 1H-13C heteronuclear chemical shift correlated spectroscopy. Alteration of the in-plane symmetry of the electronic structure of haem induced by the ligation of proximal histidyl imidazole spreads the haem carbon resonances to 32 ppm at 22 degrees C, indicating the sensitivity of those resonances to the haem electronic/molecular structure. Those resonances are potentially powerful probes in characterizing the nature of haem electronic structure.

Heme methyl hyperfine shift pattern as a probe for determining the orientation of the functionally relevant proximal histidyl imidazole with respect to the heme in hemoproteins.

Heme methyl 1H and 13C resonances of met-cyano form of myoglobin from the shark, Geleorhinus japonicus (GJMbCN), have been assigned via 1H-13C heteronuclear shift correlated spectroscopy (COSY) connectivities and their hyperfine shifts were compared with those of the corresponding resonances of some hemoproteins. Variation of the heme methyl 1H hyperfine shift pattern correlates well with the angle (phi) between the projection of the proximal histidyl imidazole plane onto the heme plane and the NII-Fe-NIV vector. The alteration of the interaction of the heme peripheral side-chains and/or the iron-bound ligand with the surrounding amino acid residues cannot account for large differences in the shifts of the corresponding heme methyl resonances between GJMbCN and sperm whale MbCN. Since the heme methyl 1H shifts for GJMbCN fall in between those of the corresponding resonances for sperm whale Mb and Aplysia limacina Mb in which the phi values have been reported to be 19 degrees and 29 degrees, respectively, the phi value in GJMb is estimated to be slightly larger than 19 degrees.

Strong vibronic coupling in heme proteins.

We report the near infrared absorption spectra of cyanomethemoglobin and cyanometmyoglobin in two different solvents (deuterated solutions containing 65% v/v glycerol(OD)3 or 65% v/v ethylene glycol(OD)2). At 25 K the spectra show a clearly resolved fine structure that can be accounted for by considering a strong coupling of the porphyrin-to-iron charge transfer transitions with a single vibrational mode at 365 cm-1. The coupling constants depend on both the specific electronic transition and the protein surrounding the chromophore, indicating once more the specificity of heme globin interactions.

The structure of the cytochrome a3-CuB site of mammalian cytochrome c oxidase as probed by MCD and EPR spectroscopy.

The nature of the complexes formed between cytochrome c oxidase and the three inhibitory ligands N3-, CN-, and S2- have been investigated by a combination of MCD and EPR spectroscopy. CN- forms a linear bridge between the Fe III a3 and CuB II, suggesting that the distance between these centers in the oxidized enzyme is between 5 and 5.25 A. This distance is too short to permit N3- to form a linear bridge and the evidence suggests this to be bent. In contrast S2- or SH- is unable to form any bridge and it seems likely that two SH- ions are bound by the bimetallic site, one to Fe III a3 and the other to CuB I. The significance of the a3-CuB distance in terms of oxygen binding and reduction is discussed.

Limits of cryofixation as seen by Fourier transform infrared spectra of metmyoglobin azide and carbonyl hemoglobin in vitrified and freeze-concentrated aqueous solution.

The limits of cryofixation were probed by investigating metmyoglobin azide and carbonyl hemoglobin in approximately 5 wt% aqueous solution by Fourier transform infrared spectroscopy. Spectra of solutions cooled slowly and recorded in steps between 295 K and 190 K are compared with those obtained by "hyperquenching" either into their glassy states at 80 K, or into freeze-concentrated solution at 170 K. For metmyoglobin azide we conclude from an analysis of its covalently and ionically bound azide that it is impossible to freeze-in its high-spin/low-spin equilibrium even by hyperquenching, and that its vitrified state must correspond to a temperature T < 226 K for the Fe(II) site of the protein. In the amide I spectral region of carbonyl hemoglobin (HbCO), a band at approximately 1654 cm-1 due to alpha-helical structures is the dominant band in spectra recorded at ambient temperature and in the vitrified state, but in the spectrum of HbCO quenched at similar rates into a freeze-concentrated state, a band at approximately 1650 cm-1, tentatively assigned to unordered structures, becomes the dominant feature. This band is absent in the spectra of freeze-concentrated samples obtained by heating a vitrified sample to 170 K. We surmise that HbCO is dehydrated by freeze-concentration to a larger extent in solution quenched rapidly at 170 K than in a vitrified solution heated to 170 K, and that this dehydration is the primary cause for HbCO's perturbation. We conclude that freeze-concentration induced by heating a vitrified solution can cause less perturbations of a protein than does quenching into a freeze-concentrated state. Therefore it can be advantageous for the practice of freeze-etching to vitrify first a solution by "hyperquenching" and thereafter freeze-etch at e.g. approximately 170 K.

XANES of carboxy and cyanomet-myoglobin. The role of the distal histidine in the bent Fe-C-O configuration.

The ligand bonding geometry of carboxy- and cyanomet-myoglobin (MbCO and MbCN) has been measured by the XANES method (X-ray Absorption Near Edge Structure). A comparison between the ligand bonding geometry of carboxy- and cyanomet-myoglobin and of chelated protoheme methyl ester shows that the bent Fe-C-O configuration is the same in both systems. Therefore, we suggest that this configuration is not associated with any steric constraint imposed by the side chains of the aminoacid residues at the distal side of the heme pocket.

Tracer diffusion of globular proteins in concentrated protein solutions.

The diffusion of tracer proteins at low concentration was measured in solutions containing "background" proteins at concentrations of up to 200 g/liter. The fractional reduction of the diffusion coefficient of tracer in the presence of a given weight/volume concentration of background species generally increases with increasing size of tracer species and with decreasing size of background species. The dependence of the diffusion constants of three out of four tracer species upon the concentrations of four background species is accounted for semiquantitatively by a simple hard particle model. Extrapolation of model calculations to higher background concentrations suggests that in solutions containing proteins at concentrations comparable to those found in biological fluid media, the diffusive transport of larger proteins and aggregates may be slower than in dilute solution by several orders of magnitude.

A quantitative J-correlation experiment for the accurate measurement of one-bond amide 15N-1H couplings in proteins.

Very precise measurements of 1JNH couplings have been made for approximately 40% of the amide sites in cyanometmyoglobin using two different experimental approaches. The first approach is a previously described frequency-based method in which the couplings are observed as splittings in the frequency-domain spectrum. The second is a new approach, along the lines of quantitative J-correlation spectroscopy, in which the coupling is encoded in the resonance intensity. Measurements obtained from the two experimental techniques are in agreement to a high degree of precision (s.d. of 0.17 Hz) and residual deviations appear to be largely random. The new method offers substantial time savings when resonances are widely dispersed in the frequency domain and may offer improved precision in these instances.

NMR evidence for slow collective motions in cyanometmyoglobin.

Residual dipolar couplings observed in NMR spectra at very high magnetic fields have been measured to a high degree of accuracy for the paramagnetic protein cyanometmyoglobin. Deviations of these measurements from predictions based on available crystallographic and solution structures are largely systematic and well correlated within a given helix of this highly alpha-helical protein. These observations can be explained by invoking collective motion and small displacements of representative helices from their reported average positions in the solid state. Thus, the measurements appear to be capable of providing important insights into slower, collective protein motions, which are likely to be important for function, and which have been difficult to study using established experimental techniques.