Structure and ligand binding properties of myoglobins reconstituted with monodepropionated heme: functional role of each heme propionate side chain

Two heme propionate side chains, which are attached at the 6 and 7 positions of the heme framework, are linked with Arg45 and Ser92, respectively, in sperm whale myoglobin. To evaluate the role of each propionate, two kinds of one-legged hemins, 6-depropionated and 7-depropionated protohemins, were prepared and inserted into the apomyoglobin to yield two reconstituted proteins. Structural data of the reconstituted myoglobins were obtained via an X-ray crystallographic analysis at a resolution of 1.1-1.4 A and resonance Raman spectroscopy. It was found that the lack of the 6-propionate reduces the number of hydrogen bonds in the distal site and clearly changes the position of the Arg45 residue with the disrupting Arg45-Asp60 interaction. In contrast, the removal of the 7-propionate does not cause a significant structural change in the residues of the distal and proximal sites. However, the resonance Raman studies suggested that the coordination bond strength of the His93-Fe bond for the protein with the 7-depropionated protoheme slightly increases compared to that for the protein with the native heme. The O2 and CO ligand binding studies for the reconstituted proteins with the one-legged hemes provide an important insight into the functional role of each propionate. The lack of the 6-propionate accelerates the O2 dissociation by ca. 3-fold compared to those of the other reconstituted and native proteins. The lack of the 7-propionate enhances the CO affinity by 2-fold compared to that of the protein with the native heme. These results indicate that the 6-propionate clearly contributes to the stabilization of the bound O2, whereas the 7-propionate plays an important role in the regulation of the Fe-His bond.     

Proton NMR study of the cyanide metmyoglobin reconstituted with meso-tetraalkylhemins. Dynamic free rotation of the synthetic hemins in the heme pocket

Incorporation of the three synthetic hemins, Fe(III) meso-tetraalkylporphyrins with the methyl, ethyl, or n-propyl groups, into apomyoglobin was followed by spectrophotometry, and the stoichiometric complex formation was confirmed. The reconstituted myoglobins bind with an equimolar amount of cyanide to exhibit visible absorption peaks at 419, 570, and 608 nm. The spectral feature was independent of the cyanide concentrations. Proton NMR spectra of the cyanide complexes resolved the pyrrole-proton signals of the hemins in a -5 to -15-ppm region, which is comparable with that of the corresponding signals of deuterohemin-containing low-spin methemoproteins. These spectral observations indicate the presence of the NC-Fe-N(His-F8) structure in the presently reconstituted cyanide metmyoglobins. The pyrrole-proton NMR signals of the hemins in cyanide metmyoglobins appeared as a singlet, doublet, or quartet for the methyl, ethyl, or n-propyl hemin complexes, respectively. The systematic NMR spectral changes suggest the dynamic free rotation of the alkylhemins about the Fe-N(His-F8) bond. Temperature-dependent NMR spectral transition of the meso-tetraethylhemin-reconstituted myoglobin was consistent with thermally regulated dynamic free rotation of the hemin in the myoglobin heme pocket.     

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.     

1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl α-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole π bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

Identification of the titrating group in the heme cavity of myoglobin. Evidence for the heme-protein pi-pi interaction

The pH dependence of the proton NMR chemical shifts of met-cyano and deoxy forms of native and reconstituted myoglobins reflects a structural transition in the heme pocket modulated by a single proton with pK 5.1-5.6. Comparison of this pH dependence of sperm whale and elephant myoglobin and that of the former protein reconstituted with esterified hemin eliminates both the distal histidine as well as the heme propionates as the titrating residue. Reconstitution of sperm whale met-cyano myoglobin with hemin modified at the 2,4-positions leads to a systematic variation in the pK for the structural transition, thus indicating the presence of a coupling between the titrating group and the heme pi system. The results are consistent with histidine FG3 (His-FG3) being the titrating group, and a donor-acceptor pi-pi interaction between its imidazole and the heme is proposed.     

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.     

Structural analysis of fish versus mammalian hemoglobins: effect of the heme pocket environment on autooxidation and hemin loss

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.     

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin.

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NH's of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.     

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].     

Peroxidase activity of hemoglobin, modified at the carboxylic groups of heme and amino acids]

The effects of modification of heme carboxylic groups by omega-aminoenantic acid and L-phenylalamine on the peroxidase activity of hemoglobin were studied. For this purpose the peroxidase activities of the original compounds--hemin, hemin-aminoenantic acid, hemin-phenylalanine and hemoglobins prepared from the hemin and globin compounds--hemoglobin, aminoenantyl-hemoglobin and phenylalanine hemoglobin--were determined. The dependence of the peroxidase activity of these compounds on their concentrations and pH was analyzed. It was shown that 40--50% modification of the heme carboxylic groups by amino acids decreases the peroxidase activity of the modified hemins and that of modified hemoglobins reconstructed from these hemins and globin. A decrease of the catalytic activity of the hemoglobin derivatives is due to a lower peroxidase activity (as compared to hemin) of the modified hemins. It is thus concluded that the amino acid modification of the carboxylic groups of heme does not affect the heme-protein interactions in the hemoglobin molecule.     

Effect of modification of heme propionate groups on the reactivity of horseradish peroxidase

Artificial horseradish peroxidases were prepared containing hemin in which propionate groups at the 6,7-positions were modified. All of the unnatural molecules had the chemical and enzymic properties of the native enzymes but not to the same extent. This finding eliminates the possibility that a propionate group in the 6- or 7-position of the hemin plays a catalytic role in compound I formation. The main effect of modifications at the positions of heme carboxyl groups may be caused by changes in the electric charge at the periphery of the hemin.     

Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.     

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)     

Horse heart myoglobin reconstituted with a symmetrical heme. A circular dichroism study

Proton NMR studies on myoglobins and hemoglobins reconstituted with non-natural hemes, possessing different side chains in the pyrrolic rings, have provided interesting information for the understanding of the mechanism governing heme reorientation in the globin pocket, during synthesis of the native protein in vivo or in the reconstitution process in vitro. More recently, circular dichroism (CD) studies have been reported as a qualitative, alternative tool, with respect to 1H-NMR for detecting heme disorder in a reconstituted myoglobin or hemoglobin. In this paper, a CD study is reported on the reconstitution of horse heart myoglobin with protoheme XIII, a heme possessing true rotational symmetry about its alpha, gamma-meso axis. The results obtained show that the reconstitution product with this heme, which binds to the apoprotein with high affinity, not dissimilar from that of the natural heme, is characterized by a CD spectrum with bands possessing rotational strengths much lower than in the native protein. Furthermore, the CD changes detected as a function of time, during heme reorientation, in the case of natural heme, are absent when the apoprotein is reconstituted with protoheme XIII. These data provide independent evidence for reorientation of the natural heme, which follows its insertion into the protein matrix.     

Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the alpha-gamma axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the alpha-gamma axis without leaving the "protein cage."     

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg-Teller approximation. This theoretical approach makes use of the crude Born-Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands for both Condon and non-Condon vibrational modes. In this framework, an x-y splitting of the Q transition for native and deuteroheme reconstituted horse myoglobin is clearly assessed and attributed to electronic perturbations that, in turn, are caused by a reduction of the typical D(4h) symmetry of the system due to heme distortions of B(1g)-type symmetry and/or to an x-y asymmetric position of the nicotinate ring; in deuteroheme reconstituted horse myoglobin the asymmetric heme peripheral substituents add to the above effect(s). On the contrary, in leghemoglobin-a no spectral splitting upon nicotinate binding is observed, pointing to a planar heme configuration in which only distortions of A(1g)-type symmetry are effective and to which the nicotinate ring is bound in an x - y symmetric position. The local dynamic properties of the heme pocket of the three proteins are investigated through the temperature dependence of spectral line broadening. Leghemoglobin-a behaves as a softer matrix with respect to horse myoglobin, thus validating the hypothesis of a looser heme pocket conformation in the former protein, which allows a nondistorted heme configuration and a symmetric binding of the bulky nicotinate ligand.     

Stability enhancement of cytochrome c through heme deprotonation and mutations

The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.     

Myoglobin structure and regulation of solvent accessibility of heme pocket

The effects of heme removal on the molecular structure of tuna and sperm whale myoglobin have been investigated by comparing the solvent accessibility to the heme pocket of the two proteins with that of the corresponding apoproteins. Although the heme microenvironment of tuna myoglobin is more polar than that of sperm whale myoglobin, the accessibility of solvent to heme is identical in the two proteins as revealed by thermal perturbation of Soret absorption. The removal of heme produces loss of helical folding and increase of solvent accessibility but the effects are rather different for the two proteins. More precisely, the loss of helical structure upon heme removal is 50% for tuna myoglobin and 15% for sperm whale myoglobin; moreover, the solvent accessibility of the heme pocket of tuna apomyoglobin is 2-3-fold greater than that of sperm whale apomyoglobin. These results have been explained in terms of the lack of helical folding in segment D, the structural organization of which may have a relevant effect in regulating the accessibility of ligands to the heme. The effects produced by charged quenchers reveal that the ligand path from the surface of the molecule to the ion atom of the heme involves a positively charged residue which may reasonably be identified as Arg-45 (sperm whale myoglobin) or Lys-41 (tuna myoglobin) on the basis of recent X-ray crystallographic information.     

High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.