1H-NMR study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin

Two-dimensional 1H NMR spectroscopy over a range of temperature through thermal unfolding has been applied to the low-spin, ferric cyanide complex of myoglobin from Aplysia limacina to search for intermediates in the unfolding and to characterize the effect of temperature on the magnetic properties and electronic structure of the heme iron. The observation of strictly linear behavior from 5 to 80 C degrees through the unfolding transition for all hyperfine-shifted resonances indicates the absence of significant populations of intermediate states to the cooperative unfolding with Tm approximately 80 degrees C. The magnetic anisotropies and orientation of the magnetic axes for the complete range of temperatures were also determined for the complex. The anisotropies have very similar magnitudes, and exhibit the expected characteristic temperature dependence, previously observed in the isoelectronic sperm whale myoglobin complex. In contrast to sperm whale Mb, where the orientation of the magnetic axis was completely temperature-independent, the tilt of the major magnetic axis, which correlates with the Fe-CN tilt, decreases at high temperature in Aplysia limacina Mb, indicating a molecular structure that is conserved with temperature, although more plastic than that of sperm whale Mb. The pattern of contact shifts reflects a conserved Fe-His(F8) bond and pi-spin delocalization into the heme, as expected for the orientation of the axial His imidazole.     

Crystal structure of ferric Aplysia limacina myoglobin at 2 X 0 A resolution

The three-dimensional structure of ferric myoglobin from the mollusc Aplysia limacina has been refined at 2 X 0 A resolution. The crystallographic R factor, calculated at this stage, is 0 X 194. Despite its high content of apolar residues (both aromatic and aliphatic), Aplysia limacina myoglobin, which contains only one histidine residue (at the proximal position), has a structure that conforms to the common eight-helices globin fold observed in other phyla.     

Binding mode of azide to ferric Aplysia limacina myoglobin. Crystallographic analysis at 1.9 A resolution

The binding mode of azide to the ferric form of Aplysia limacina myoglobin has been studied by X-ray crystallography. The three-dimensional structure of the complex has been refined at 1.9 A resolution to a crystallographic R-factor of 13.9%, including 126 ordered solvent molecules. Azide binds to the heme iron, at the sixth co-ordination position, and is oriented towards the outer part of the distal site crevice. This orientation is stabilized by an ionic interaction with the side-chain of Arg66 (E10) which, from an outer orientation in the 'aquo-met' ligand-free myoglobin, folds back towards the distal site in the presence of the anionic ligand. In the absence of a hydrogen bond donor residue at the distal E7 position in Aplysia limacina myoglobin, a different polar residue, Arg66 at the E10 topological position, has been selected by molecular evolution in order to grant ligand stabilization.     

X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin at 2.0 A resolution. Stabilization of the fluoride ion by hydrogen bonding to Arg66 (E10)

The X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin has been solved and refined at 2.0 A resolution; the crystallographic R-factor is 13.6%. The fluoride ion binds to the sixth co-ordination position of the heme iron, 2.2 A from the metal. Binding of the negatively charged ligand on the distal side of the heme pocket of this myoglobin, which lacks the distal His, is associated with a network of hydrogen bonds that includes the fluoride ion, the residue Arg66 (E10), the heme propionate III, three ordered water molecules and backbone or side-chain atoms from the CD region. A comparison of fluoride and oxygen dissociation rate constants of A. limacina myoglobin, sperm whale (Physeter catodon) myoglobin and Glycera dibranchiata monomeric hemoglobin, suggests that the conformational readjustment of Arg66 (E10) in A. limacina myoglobin may represent the molecular basis for ligand stabilization, in the absence of a hydrogen-bond donor residue at the distal E7 position.     

Heme protein fluorescence versus pressure.

Fluorescence spectra of several ferric heme proteins have been measured vs. pressure to 6,000 bars. Sperm whale myoglobin (SW Mb), Aplysia myoglobin, leghemoglobin (Lb), and cytochrome P450 all show excitation and emission spectra characteristic of tryptophan in proteins with peak emission at 330-340 nm. At one bar, the fluorescence is weak due to energy transfer to the heme group, which makes the yield a sensitive probe of protein unfolding at high pressure. After an initial decrease of a few percent per kbar, the protein shows a large increase in fluorescence at high pressure. The increase is pH dependent and the results indicate that several high pressure states occur. For SW Mb at 15 degrees C an increase of a factor of 20 occurs with midpoint at 2,000 bars at pH 5 and is only partially reversible, while the increase at pH 7 occurs at 4,000 bars and is only half as large and is completely reversible. Aplysia Mb and Lb show a similar effect, but unfold at a higher pressure than SW Mb. P450 also shows a transition to a state of higher fluorescence, but the transition in this case is irreversible as a stable form, P420, is formed. The fluorescence intensity measurements permit an estimation of the increase in the TRY-heme distance in the high pressure state.     

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.     

Isocyanide binding kinetics to monomeric hemoproteins. A study on the ligand partition between solvent and heme pocket.

The kinetics of methyl-, ethyl-, iso-propyl-, and ter-butyl-isocyanide binding to Aplysia limacina myoglobin (distal His----Lys) and the isolated beta chains from hemoglobin Zurich (distal His----Arg) have been investigated by flash photolysis at various temperatures above 0 degrees C. Sperm whale (Physter catodon) myoglobin and the isolated beta chains from normal adult hemoglobin have been used as references. In most reaction systems investigated the apparent extent of photolysis increases with temperature. For sperm whale myoglobin and the normal beta chains the increase is of the same magnitude and not correlated to the type of ligand used. On the contrary, for the two proteins lacking the distal histidine, the phenomenon is dependent on the size of the alkyl side chain of the ligand. The results, analyzed on the basis of the multibarrier model (Austin, R.H., K.W. Beeson, L. Eisenstein, H. Frauenfelder, and I.C. Gunsalus, 1975, Biochemistry, 16:5355-5373), suggest that the partition of the ligand molecules between the solvent and the heme pocket, occurring during the photolysis process, is primarily determined by interactions between the ligand and residues in the heme cavity rather than by diffusion through the protein matrix.     

Haem disorder in two myoglobins: comparison of reorientation rate.

The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237, 613-616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon.     

Construction and characterization of a chimeric myoglobin

In order to investigate the functional and structural role of modular structure in globins, we have engineered a chimeric myoglobin (ChimMb) in which the first and third exon come from the gene coding for the sperm whale Mb and the second exon from the gene coding for Aplysia limacina Mb. This ChimMb, fused to the Maltose Binding Protein (MBP) and expressed in Escherichia coli as an apoprotein, binds protoheme in a 1:1 stoichiometric ratio. Based on some functional and spectroscopic properties, we conclude that the central core of the ChimMb (which derives from A. limacina) is native-like. On the other hand, the ChimMb deprived (by proteolytic digestion) of the fused MBP displays a considerably reduced stability. These results suggest that the sperm whale A-G-H nucleus does not contribute significantly to the overall stability of the ChimMb.     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.     

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].     

Thermodynamics of the reaction of ferric myoglobin from Aplysia limacina with azide and fluoride. Dependence of enthalpy changes on pH

The effect of pH on the enthalpy changes for binding of azide and fluoride to ferric myoglobin from Aplysia limacina, which lacks the distal histidine, has been investigated. Over the whole pH range explored (3.8 to 9.5), -delta H degrees values for the formation of the hemoprotein-ligand complexes are: (1) much greater than the variations in -delta G degrees; (2) always negative; and (3) show a dependence upon pH characterized by a maximum for azide and a minimum for fluoride binding, centered at pH 4.55 (identical to pHch). This value agrees well with that expected from the linear correlation between pHch and the simple function "Lys+Arg-Glu-Asp-2" proposed by Beetlestone and others. Data reported here greatly extend the pH range for which the linear correlation between the net charge of the macromolecule and pHch has been found to hold, and indicate unequivocally that the pH dependence of -delta H degree for the binding of anionic ligands does not uniquely require the presence of the histidyl residue at the distal position.     

Kinetics of carbon monoxide binding to monomeric hemoproteins. Role of the proximal histidine

The effect of pH on (i) the second-order rate constant for CO binding and (ii) the spectral properties of the deoxygenated derivative of several monomeric hemoproteins has been investigated in the pH range between 2.3 and 9.0. As in the case of 3-[1-imidazolyl]-propylamide monomethyl ester mesoheme, the rate constant for CO binding to sperm whale, horse, Dermochelys coriacea, Coryphaena hippurus, and Aplysia limacina myoglobins (the latter only in the presence of acetate/acetic acid mixture) increases, as the pH is lowered, to a value at least 1 order of magnitude higher than at pH 7.0. Such an effect is not observed in A. limacina myoglobin (in the absence of the acetate/acetic acid mixture) and Chironomus thummi thummi erythrocruorin. Moreover, the absorption spectrum, in the visible region, of the deoxy derivative of all these monomeric hemoproteins (with the exception of A. limacina myoglobin in the absence of the acetate/acetic acid mixture) undergoes a transition as the pH is lowered, an effect observed previously with 3-[1-imidazolyl]-propylamide monomethyl ester protoheme. On the basis of analogous spectroscopic and kinetic properties of chelated heme model compounds we attribute this behavior to the protonation of the N epsilon of the proximal imidazole involved in the bond with the iron atom. On the basis of this model the movement of the iron atom to the heme plane appears as a crucial step for CO binding, the activation free energy of the process amounting to approximately 2 kcal/mol.     

Engineering His(E7) affects the control of heme reactivity in Aplysia limacina myoglobin

Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 A crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.     

Histidine and not tyrosine is required for the peroxide-induced formation of haem to protein cross-linked myoglobin

Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 - 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr(103) --> Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome.     

Structural determinants of fluoride and formate binding to hemoglobin and myoglobin: crystallographic and 1H-NMR relaxometric study.

The x-ray crystal structure of the fluoride derivative of ferric sperm whale (Physeter catodon) myoglobin (Mb) has been determined at 2.5 A resolution (R = 0.187) by difference Fourier techniques. The fluoride anion, sitting in the central part of the heme distal site and coordinated to the heme iron, is hydrogen bonded to the distal His(64)E7 NE2 atom and to the W195 solvent water molecule. This water molecule also significantly interacts with the same HisE7 residue, which stabilizes the coordinated fluoride ion. Moreover, fluoride and formate binding to ferric Aplysia limacina Mb, sperm whale (Physeter catodon) Mb, horse (Caballus caballus) Mb, loggerhead sea turtle (Caretta caretta) Mb, and human hemoglobin has been investigated by 1H-NMR relaxometry. A strong solvent proton relaxation enhancement is observed for the fluoride derivatives of hemoproteins containing HisE7. Conversely, only a small outer-sphere contribution to the solvent relaxation rate has been observed for all of the formate derivatives considered and for the A. limacina Mb:fluoride derivative, where HisE7 is replaced by Val.     

Solution (1)H NMR study of the influence of distal hydrogen bonding and N terminus acetylation on the active site electronic and molecular structure of Aplysia limacina cyanomet myoglobin

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution (1)H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small approximately 2 degrees rotation of the axial His in rWT relative to true WT and a approximately 3 degrees rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.     

Equilibrium and kinetic evidence for a transition between six- and five-coordinate ferrous heme in the nitric oxide derivative of Aplysia myoglobin

The pH dependence in the range 3--7 of the optical absorption and electron paramagnetic resonance of the nitric oxide adduct of ferrous Aplysia myoglobin is reported. Optical spectra in the Soret region show a transition between two conformers with an apparent pK in the range 3.5--5 depending on the presence of carboxylic anions as third component. In the same pH range, the EPR spectrum undergoes a change from a 9-line to a 3-line hyperfine pattern in the g. region, similar to that reported for synthetic heme derivatives and for other hemoproteins. The structural interpretation of the pH-induced transition experienced by Aplysia myoglobin nitric oxide is that of a proton-linked cleavage of the proximal bond as suggested by several lines of evidence. Temperature-jump measurements allowed an estimation of the relaxation time for the process, which is of the order of 0.3 ms at 25 degrees C.     

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)