Crystal structures of modified myoglobins. II. Relation between oxygen affinity properties and structural changes around heme in myoglobins reconstituted with 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme

The crystal structures of sperm whale metmyoglobins reconstituted with three kinds of modified hemes, 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.216, 0.219, and 0.195, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The 2-vinyl-4-isopropyldeuteroheme was found to be in a reverse orientation, in which the heme plane is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other two hemes were the same as that of protoheme in native myoglobin. In the myoglobins with 2,4-diisopropyldeuteroheme and 2-vinyl-4-isopropyldeuteroheme, both of which have lower oxygen affinities than native myoglobin, the bulky isopropyl side chain pushes Phe 43 0.7 A toward His 64 (the distal histidine) in the former, and the whole E helix at most 1.5 A, including a 0.7 A shift of the His 64 imidazole ring, in the latter. The changes of the structures prevent His 64 from forming a hydrogen bond with the liganded oxygen molecule, so that these two modified myoglobins show low oxygen affinities. On the other hand, there is no such drastic displacement in myoglobin with 2-isopropyl-4-vinyldeuteroheme, which has a slightly higher oxygen affinity than native myoglobin.     

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.     

Resonance Raman assignment and evidence for noncoupling of individual 2- and 4-vinyl vibrational modes in a monomeric cyanomethemoglobin

We have investigated the resonance Raman spectra of monomeric insect cyanomethemoglobins (CTT III and CTT IV) reconstituted with (1) protohemes IX selectively deuterated at the 4-vinyl as well as the 2,4-divinyls, (2) monovinyl-truncated hemes such as pemptoheme (2-hydrogen, 4-vinyl) and isopemptoheme (2-vinyl, 4-hydrogen), (3) symmetric hemes such as protoheme III (with 2- and 3-vinyls) and protoheme XIII (with 1- and 4-vinyls), and (4) hemes without 2- and 4-vinyls such as mesoheme IX, deuteroheme IX, 2,4-dimethyldeuteroheme IX, and 2,4-dibromodeuteroheme IX. Evidence is presented that the highly localized vinyl C = C stretching vibrations at the 2- and 4-positions of the heme in these cyanomet CTT hemoglobins are noncoupled and inequivalent; i.e., the 1631- and 1624-cm-1 lines have been assigned to 2-vinyl and 4-vinyl, respectively. The elimination of the 2-vinyl (in pemptoheme) or the 4-vinyl (in isopemptoheme) does not affect the C = C stretching frequency of the remaining vinyl. Furthermore, two low-frequency vinyl bending modes at 412 and 591 cm-1 exhibit greatly different resonance Raman intensities between 2-vinyl and 4-vinyl. The observed intensity at 412 cm-1 is primarily derived from 4-vinyl, whereas the 591-cm-1 line results exclusively from the 2-vinyl. Again, there is no significant coupling between 2-vinyl and 4-vinyl for these two bending modes.     

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.     

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.     

Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide.

In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.     

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.     

Preparation of artificial 2-, 3-, 4- and 8-domain myoglobins and comparison of their autoxidation rates

Although most hemoglobins and myoglobins consist of 15-kDa single-domain subunits, structurally unusual hemoglobins, such as Artemia 9-domain and Barbatia 2-domain hemoglobins, occur naturally in several invertebrates. These hemoglobins appear to be the result of gene duplication and fusion. Using cDNA coding for the open reading frame of Aplysia kurodai myoglobin, artificial cDNA inserts corresponding to contiguous dimer, trimer, tetramer and octamer myoglobins (2-, 3-, 4- and 8-domain myoglobins) were prepared and cloned into pMAL or pQE plasmids. These artificial myoglobins and wild-type single-domain myoglobins were successfully expressed in Escherichia coli in the heme-attached, oxygenated form. Myoglobin was purified partially by ammonium sulfate fractionation and gel filtration, and autoxidation rates were examined. The autoxidation rates of recombinant wild-type myoglobins with MBP or hexameric His tag were comparable to those of native myoglobin, suggesting that the recombinant proteins appear to be properly folded and that the N-terminal MBP or His tag does not have an affect on the rate. On the other hand, the rates were significantly decreased in the 2- and 3-domain myoglobins (50% and 30% of the single-domain myoglobins, respectively). The rates for 4- and 8-domain myoglobins were similar to those for 3-domain myoglobin. These results indicate that the artificial poly-domain structure of myoglobin is more stable than the usual single-domain myoglobin from the viewpoint of storage of bound dioxygen.     

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.     

Oxygenation and EPR spectral properties of Aplysia myoglobins containing cobaltous porphyrins.

Cobalt myoglobins (Aplysia) have been reconstituted from apo-myoglobin (Aplysia) and proto-, meso-, and deutero-cobalt porphyrins. Each of them showed the 30--60 times lower oxygen affinity than those of the corresponding cobalt myoglobins (Sperm whale). Kinetic investigation of their oxygenation by the temperature-junp relaxation technique showed that the low oxygen affinity of cobalt myoglobin (Aplysia) is due to a large dissociation rate constant. the electron paramagnetic resonance (EPR) spectrum of oxy cobalt myoglobin (Aplysia) is affected by the replacement of H2O with D2O, suggesting a possible interaction between the bound oxygen and the neighboring hydrogen atom. A low temperature photodissociation study showed that the product of photolysis of oxy cobalt myoglobin (Aplysia) gives an EPR spectrum different from that of the deoxy-cobalt myoglobin (Aplysia) and from that of the photolysed form of oxy-cobalt myogloin (Sperm whale). These observations suggest that in oxy-cobalt myoglobin (Aplysia) the bound oxygen might interact with amino acid adjacent to it, but the interaction is weaker than that in oxy cobalt myoglobin (Sperm whale).     

Decrease in oxygen affinity of myoglobin by formylation of vinyl groups of heme.

Three kinds of green synthetic myoglobin were prepared by recombination of horse heart apomyoglobin with spirographis (2-formyl-4-vinyl-), isospirographis (2-vinyl-4-formyl-), and 2,4-diformyldeuterohemins. The optical and oxygen binding properties of the reconstituted myoglobins containing two isomeric monoformyl-monovinylhemins were found to be different. The oxygen affinities (P50) of spirographis and 2,4-diformylmyoglobins are 2.7 and 2.8 mm Hg, respectively, at 25 degrees, and about 2.5 times lower than that of native protomyglobin, while that of isospirographis myoglobin is 1.0 mm Hg and is similar to native myoglobin. Spirographis oxymyoglobin has absorption maxima (alpha, beta, and Soret bands) at 601, 556.5, and 435 nm, isospirographis oxymyoglobin at 595, 550, 429 nm, and 2,4-diformyl oxymyoglobin at 603, 563.5, and 447 nm. The optical red shifts as well as the decrease in the oxygen affinities of these myoglobins are attributed mainly to the presence of strongly electron-attractive formyl side chains. Since the free isomers of monoformyl-monovinyl heme have similar properties, the differences observed after recombination with apoprotein must be caused by interactions with apomyoglobins. The degree of such a protein effect may be estimated by comparing the absorption spectra of heme before and after recombination and was found to differ among the various myoglobins. Comparison of the oxygen affinities of the myoglobins taking account of this protein factor showed that the increase in the P50 values are inversely related to that in the pK3 values of the free porphyrins. These results suggest the involvement of pi bonding in determining the oxygen-iron bond strength.     

Comparative analysis of the spatial organization of myoglobins. I. Hydrophobicity profiles]

Hydrophobicity profiles of myoglobins in the animal species far remote in the evolutionary series are considerably similar. A complete coincidence as to the arrangement of hydrophobic zones along the polypeptide chain in myoglobins of the compared species (from a man to mollusc) is revealed at the beginning of alpha-helix of B-segment and in the area corresponding to a cluster which embodies a heme- bound water molecule, distal histidine E7 being directed to this cluster. The mollusc myoglobin with two absent (as compared to myoglobins of other species) hydrophobic sites differs in the profile of hydrophobicity most of all. It is supposed that hydrophobic nuclei forming the heme circumference create a globule "skeleton" thus pre-setting general spatial structure of the myoglobin molecule, which is very significant for its functional activity.     

Heme orientational disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional consequences

A ring fluorinated heme, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2-fluoro-7,12,18-trimethyl-porphyrinatoiron(III), has been incorporated into human adult hemoglobin (Hb A). The heme orientational disorder in the individual subunits of the protein has been readily characterized using (19)F NMR and the O(2) binding properties of the protein have been evaluated through the oxygen equilibrium analysis. The equilibrated orientations of hemes in alpha- and beta- subunits of the reconstituted protein were found to be almost completely opposite to each other, and hence were largely different from those of the native and the previously reported reconstituted proteins [T. Jue, G.N. La Mar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy, Biochem. Biophys. Res. Commun. 119 (1984) 640-645]. Despite the large difference in the degree of the heme orientational disorder in the subunits of the proteins, the O(2) affinity and the cooperativity of the protein reconstituted with 2-MF were similar to those of the proteins reconstituted with a series of hemes chemically modified at the heme 3- and 8-positions [K. Kawabe, K. Imaizumi, Z. Yoshida, K. Imai, I. Tyuma, Studies on reconstituted myoglobins and hemoglobins II. Role of the heme side chains in the oxygenation of hemoglobin, J. Biochem. 92 (1982) 1713-1722], whose O(2) affinity and cooperativity were higher and lower, respectively, relative to those of native protein. These results indicated that the heme orientational disorder could exert little effect, if any, on the O(2) affinity properties of Hb A. This finding provides new insights into structure-function relationship of Hb A.     

Kinetic study of CO and O2 binding to horse heart myoglobin reconstituted with synthetic hemes lacking methyl and vinyl side chains

Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this view.     

The structure of hemoglobin reconstituted with deuteroheme

The structure of horse methemoglobin reconstituted with deuteroheme, in which the heme vinyl substituents are replaced by hydrogens, has been compared with that of native horse methemoglobin by X-ray difference Fourier techniques. The tertiary structures of the two molecules are almost completely identical, with the exception of small perturbations in the globin which occur in direct response to the missing vinyls. These perturbations are, however, highly localized and do not propagate beyond the immediate vicinity of the hemes. Contrary to expectation, complete removal of these heme vinyls results in much less drastic structural changes than does replacement by ethyl substituents, as in methemoglobin reconstituted with mesoheme.     

Assignments of the paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin by selective deuteration

Three of the four paramagnetically shifted heme methyl nuclear magnetic resonance peaks of cyanometmyoglobin could be assigned by comparing the proton nuclear magnetic resonance spectra of myoglobins reconstituted from selectively deuterated hemes. These spectra indicate that the fourth methyl nuclear magnetic resonance peak has to be looked for outside the region ?9 to ?43 parts per million.     

Conformational free energies of myoglobins of small mammals

Myoglobins from three small placental mammals and one small marsupial were isolated from cardiac or skeletal muscle. The conformational free energies of these four myoglobins were estimated from guanidinium chloride unfolding data at pH 8 and 25 degrees. The myoglobins from rat and rabbit are more stable than that of the most stable myoglobin previously studied, that of the sperm whale. In addition, these two myoglobins unfold with greater cooperativity than previously characterized myoglobins. The data obtained herein demonstrate unequivocally for the first time that the stability of homeotherm myoglobins correlates with neither the size of the organism nor its metabolic rate.     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

Purification and characterization of the myoglobins of Paramecium tetraurelia.

1. The molecular variants of the myoglobins of Paramecium tetraurelia have been purified as five separate components and designated Mb 1 to Mb 5. 2. The five molecular forms are homogeneous on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IF). 3. The myoglobin species appear to have identical molecular weights and amino acid compositions and differ only in their isoelectric points and relative concentrations in vivo. 4. The myoglobin species have an apparent molecular weight of 15,000 +/- 500 and possess a single heme group per mole which appears to be protoporphyrin IX. 5. The amino acid composition of the five species is: lys12, his3, arg4, asp17, thr9, ser6, glu16, pro4, gly11, ala15, cys0, val8, met2, ile7, leu10, tyr5, phe7. 6. The spectra of several ferrous and ferric derivatives of the mixture Mb 1-Mb 5 are presented.     

Comparison of the biochemical and molecular properties of myoglobins from three Biomphalaria species

Myoglobin is a globin with heme as prosthetic group whose main known biological role is to bind to O2 reversibly. On account of their large diversity, globins from mollusks have contributed to the study of this protein class. The cDNA of the myoglobins from Biomphalaria straminea and Biomphalaria tenagophila, which have a glutamine as distal residue (E7), were constructed and analyzed by bioinformatic tools. Native (not recombinant) myoglobins of these two Biomphalaria species were purified and their experimental molecular mass (about 16 kDa) and pI (about (8.0) were provided. Data analysis showed that these proteins are monomers with the signature for the classic myoglobin fold and they are blocked in amino terminus probably by an acetyl group. Values of the autoxidation rates showed that these myoglobins oxidized slowly. About the primary sequences of the myoglobins, they turned out to be satisfactory to group mollusks in phylogenetic class.