Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

Dynamic analysis of efficient heme rotation in myoglobin by NMR spectroscopy.

Sperm whale myoglobin was reconstituted with 1,4,5,8-tetramethylhemin. The hyperfine-shifted proton NMR signals from the prosthetic group exhibit remarkable pattern changes around 15 degrees C, while the globin resonances are normal to obey the Curie law. The NMR anomaly specifically observed for the heme signals suggests a slow to rapid rotational transition of the hemin about the iron-histidine bond. The temperature-dependent pattern changes were quantitatively analyzed by a dynamic NMR method. Two sets of analyses with the heme-methyl and pyrrole-proton lines consistently afforded delta H not equal to = 16.3 kcal/mol, delta S not equal to = 14.0 e.u., delta G not equal to = 12.1 kcal/mol at 298 K, and a frequency of 90 degrees heme rotation 5600 s-1 at 20 degrees C. The relatively large activation entropy suggests that structural rearrangements at the direct heme vicinity are involved and that efficient heme rotation is accomplished by a number of fluctuative local heme-globin contacts within a conserved crevice structure.     

1H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b5

1H nuclear magnetic resonance spectroscopy was used to assign the hyperfine-shifted resonances and determine the position of a side chain in the heme cavity of wild-type rat apocytochrome b5 reconstituted with a series of synthetic hemins possessing systematically perturbed carboxylate side chains. The hemins included protohemin derivatives with individually removed or pairwise shortened and lengthened carboxylate side chains, as well as (propionate)n(methyl)8-nporphine-iron(III) isomers with n = 1-3 designed to force occupation of nonnative propionate sites. The resonance assignments were effected on the basis of available empirical heme contact shift correlations and steady-state nuclear Overhauser effect measurements in the low-spin oxidized proteins. The failure to detect holoproteins with certain hemins dictates that the stable holoproteins, unlike the case of myoglobin, demand the axial iron-His bonds and cannot accommodate carboxylate side chains at interior positions in the binding pocket. Hence, the heme pocket interior in cytochrome b5 is judged much less polar and less sterically accommodating than that of myoglobin. The propionate occupational preference was greatest as the native 7-propionate site, but also possible at the nonnative crystallographic 5-methyl or 8-methyl positions. Only for a propionate at the crystallographic 8-methyl position was a significant perturbation of the native molecular/electronic structure observed, and this was attributed to an alternative propionate-protein hydrogen bond at the crystallographic 8-methyl position. The structures of the transient protein complexes detected only shortly after reconstitution reveal that the initial encounter complexes during assembly of holoprotein from apoprotein and hemin involve one of the two alternate propionate-protein links at either the 7-propionate or native 8-methyl position. In a monopropionate hemin, this leads to the characterization of a new type of heme orientational disorder involving rotation about a N-Fe-N axis.     

Etiohemin as a prosthetic group of myoglobin

Sperm whale myoglobin was reconstituted with etioheme and the stoichiometric complex formation was confirmed. The proton NMR spectrum of the deoxy myoglobin exhibits an NH signal from the proximal histidine at 78.6 ppm, indicating heme incorporation into the heme pocket to form the Fe-N(His-F8) bond. The appearance of a single set of the heme-methyl NMR signals shows that etioheme without acid side-chains specifically interacts with the surrounding globin. The visible spectral data suggest retention of a normal iron coordination structure. The functional and NMR spectral properties of etioheme myoglobin are similar to those of mesoheme myoglobin, reflecting the absence of the electron-withdrawing heme vinyl groups.     

Proton NMR study of myoglobin reconstituted with 3, 7-diethyl-2, 8-dimethyl iron porphyrin: remarkable influence of peripheral substitution on heme rotation

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N-H resonance at 82.5 ppm to indicate the formation of the Fe--N (His93) bond. The pyrrole-protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between -10 and -18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole-protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.     

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.     

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.     

Nuclear magnetic resonance studies of hemoproteins. IV. Hindered rotation of heme side methyl group as a probe for studying van der Waals contacts in the heme side environments of myoglobin derivatives.

220 MHz roton NMR spectral evidence for restricted rotation of one methyl group in the heme side chain of ferric horse cyanomyoglobin is reported here. Temperature dependence of this methyl proton signal was computer-simulated, yielding 14,8 kcal/mol for the methyl hindered rotation. Ionic additives such as NaCl and (NH4) 2 minus SO4 caused a slackening of this restriction of methyl rotation, evidenced from collapse of methyl signal doubling by the addition of these ionic substances. This is discussed in terms of breaking of the salt bridge formed between one of the propionate COO minus group of heme and a part of the apoprotein which might lead to constraint of one of the heme side methyl groups. The peculiarity of hyperfine-shifted methyl proton signals for other myoglobin complexes such as azide and imidazole derivatives is also discussed briefly in terms of constraint of heme side methyl group buried in a hydrophobic cleft.     

Assignment of the ferriheme resonances of high- and low-spin forms of the symmetrical hemin-reconstituted nitrophorins 1–4 by 1H and 13C NMR spectroscopy: the dynamics of heme ruffling deformations

The four major nitrophorins (NPs) of the adult blood-sucking insect Rhodnius prolixus have been reconstituted with the "symmetrical hemin" 2,4-dimethyldeuterohemin, and their NMR spectra have been investigated as the high-spin (S = 5/2) aqua and low-spin (S = 1/2) N-methylimidazole (NMeIm) and cyanide complexes. The NMeIm complexes allow assignment of the high-spin hemin resonances by saturation transfer difference spectroscopy. The cyanide complexes were investigated as paramagnetic analogues of the NO complexes. It is shown that the hemin ring is highly distorted from planarity, much more so for NP2 than for NP1 and NP4 (with ruffling being the major distortion mode), for both high- and low-spin forms. For the cyanide complexes, the conformation of the distorted ring changes on the NMR timescale to yield chemical exchange (exchange spectroscopy, EXSY) cross peaks for NP1sym(CN), NP3sym(CN) and NP4sym(CN) but not for NP2sym(CN). These changes in nonplanar conformation are visualized as a "rolling" of the ruffled macrocycle ridges through some number of degrees, the lowest-energy ruffling mode. This probably occurs in response to slow protein dynamics that cause the I120 and L132 side chains in the distal heme pocket to move in opposite directions (up and away vs. down and toward the hemin ring). This in turn changes the out-of-plane displacements of the 2M and 3M of the symmetrical hemin on the NMR timescale. Two other types of dynamics, i.e., changes in heme seating and NMeIm rotation, are also observed. The highly distorted heme and the dynamics it causes are unique to the NPs and a few other heme proteins with highly distorted macrocycles.     

19F NMR study of protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin

 A novel C ₂-symmetric ring-fluorinated hemin, 13,17-bis(2-carboxyethyl)-2,8,12,18-tetramethyl-3,7-difluoroporphyrinatoiron(III), has been synthesized and was incorporated into sperm whale apomyoglobin to investigate protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin (Mb) using ¹⁹F NMR spectroscopy. NMR signals for ¹⁹F atoms introduced as substituents on the present heme in ferrous low-spin and high-spin and ferric low-spin complexes have been observed and their shifts sharply reflect not only the electronic nature of the heme iron, but also in-plane asymmetry of the heme electronic structure. The two-fold symmetric electronic structure of the ring-fluorinated hemin is clearly manifested in the ¹⁹F and ¹H NMR spectra of its dicyano complex. The chemical equivalence of the two fluorine atoms of the heme is removed in the active site of myoglobin and the splitting of the two ¹⁹F NMR signals provides a quantitative probe for characterizing the rhombic perturbation of the heme electronic structure induced by the heme-protein interaction. The in-plane asymmetry of heme electronic structures in carbonmonoxy and deoxy Mbs have been analyzed for the first time on the basis of the shift difference between the two ¹⁹F NMR signals of the heme and is interpreted in terms of iron-ligand binding and/or the orbital ground state of the heme. A potential utility of ¹⁹F NMR, combined with the use of a symmetric fluorinated hemin, in characterizing the heme electronic structure of myoglobin in a variety of iron oxidation, spin, and ligation states, is presented. Received: 23 December 1999 / Accepted: 3 April 2000     

Deuterium NMR study of structural and dynamic properties of horseradish peroxidase

High field deuterium NMR spectra have been recorded for various horseradish peroxidase complexes reconstituted with hemins possessing specific 2H labels. The line width of the 2H NMR signals of deuteroheme reconstituted-horseradish peroxidase (HRP) and its cyano complex for the immobilized skeletal 2-2H and 4-2H labels yield the overall protein rotational correlation time (22 ms at 55 degrees C), which is consistent with expectations based on molecular weight. Meso-2H4 labels yield broad (1.3 kHz) signals just upfield from the diamagnetic protein envelope for HRP, and in the central portion of the protein envelope for the CN- ligated resting state HRP. Meso-2H4-labeled mesohemin-reconstituted HRP exhibits a similar signal but shifted further upfield by approximately 10 ppm. The net upfield meso-H hyperfine shifts confirm a five-coordinate structure for resting state HRP. 2Ha resonances for essentially rotationally immobile vinyl groups were detected in both resting state HRP and CN- ligated resting state HRP. Heme methyl-2H-labeling yields relatively narrow lines (approximately 80 Hz) indicative of effective averaging of the quadrupolar relaxation by rapid methyl rotation. Thus the 2H line width of rapidly rotating methyls in hemoproteins can be used effectively to determine the overall protein tumbling rate. Preliminary 2H experiments in meso-2H4-labeled compound I do not support large pi spin density at these positions on the porphyrin cation radical, and argue for a a1u rather than a a2u orbital ground state.     

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.     

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)     

1H-NMR assignments and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution

Cytochrome b5 reconstituted with specifically deuterated hemins has led to the assignment of the resolved 6,7 beta-propionate protons and heme meso protons. Freshly reconstituted cytochrome b5 contains a mixture of two isomers in an approx. 1:1 ratio. As time proceeds the minor isomer decreases in intensity until the equilibrium ratio, approx. 8:1, of the two isomers is reached. The rate of the heme disorder kinetics was investigated for cytochrome b5 as a function of pH, oxidation state and 2,4 heme substitutents. Comparison of the kinetic data for cytochrome b5 with that obtained for other b-type heme proteins supports the proposal that the heme disorder arises from a 180 degree rotation of the heme about the alpha, gamma-meso axis. Computer-difference methods allow the spectra of the two individual isomers to be generated. Comparison of the NMR spectral parameters for the two individual isomers indicates small structural differences for amino acid side-chain orientations.     

The impact of altered protein-heme interactions on the resonance Raman spectra of heme proteins. Studies of heme rotational disorder

Heme proteins that have been reconstituted with certain hemins may contain substantial fractions of a minor component in which the orientation of the heme in the folded pocket differs from the major ("native") conformation by a 180 degrees rotation about the alpha-gamma meso axis. In fact, this minor component has also been shown to exist in some native proteins, including several mammalian globins. While resonance Raman spectroscopy has emerged as a powerful probe of active site structure of heme proteins, no systematic study has yet been undertaken to elucidate the specific spectral changes associated with this disorder. In the present work, combined analyses of the temporal behavior of both NMR and RR data sets have been completed to permit the extraction of a unique RR spectrum for the disoriented form, documenting rather dramatic changes associated with this rotational disorder. In addition, the use of protohemes bearing selectively deuterated peripheral methyl groups has permitted the association of the observed modes with specific fragments of the heme residing in the reversed orientation. The studies conducted here clearly illustrate the exquisite sensitivity of low frequency heme deformation modes to altered protein-heme interactions.     

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.     

High-pressure nuclear magnetic resonance studies of hemoproteins. Pressure-induced structural changes in the heme environments of ferric low-spin metmyoglobin complexes

In order to gain an insight into nonbonded interactions in the heme microenvironments of hemoproteins, proton NMR spectra of the cyanide and methylamine complexes of metmyoglobin and its derivatives reconstituted with deutero- and meso-hemins in H2O were studied under high pressures. The exchangeable NH proton of distal histidyl imidazole exhibits substantial pressure-induced shift while the proximal histidyl NH proton shows no pressure effect for the cyanide complexes. The heme peripheral proton signals, especially 5- and 8-methyl and vinyl C alpha H resonances, were also affected by pressure. These observations are interpreted as arising from pressure-induced structural changes in the heme crevice in which the pressure effects are localized to the distal side rather than the proximal side and from possible changes in the van der Waals contacts at the heme periphery with nearby amino acid residues.     

Spin-state equilibrium in the model complexes of azide hemoprotein

Addition of NaN3 to ferric protohemin biscoordinated with 1-methylimidazole (1-MeIm) or 2-methylimidazole (2-MeIm) in (CH3)2SO resulted in sizeable visible absorption changes, corresponding to the formation of the mixed ligand complexes, hemin X N-3 X 1-MeIm and hemin X N-3 X 2-MeIm. The visible absorption spectrum of the 1-MeIm complex was closely similar to those of azide hemoproteins, while the 2-MeIm derivative exhibited intensified 500 and 625 nm bands and depressed 540 and 570 nm peaks. The iron-bound N-3 of the model complexes exhibited two infrared stretching bands, which were assigned to the high- and low-spin peaks. The intensity of the high-spin infrared peaks increased at higher temperature. From the analyses of the infrared spectral changes, the thermodynamic values of the thermal spin equilibria were determined to be delta H = -3920 cal/mol and delta S = -11.1 e.u. for hemin X N-3 X 1-MeIm and delta H = -2150 cal/mol and delta S = 7.9 e.u. for hemin X N-3 X 2-MeIm. The thermodynamic values of the 1-MeIm complex are similar to the reported values for azide metmyoglobin, suggesting that the contribution from the nonbonded porphyrin-globin contacts to the spin equilibrium is small in azide metmyoglobin. Comparison of the delta H and delta S values among model systems indicates that delta H and delta S compensation similar to that observed in hemoprotein also holds in the models. This may suggest an underlying common denominator for the spin-equilibrium mechanisms in hemins and hemoproteins.     

Peroxidative crosslinking of myosins.

The effect of myoglobin, free hemin and H2O2 on myosins from heart and skeletal muscle was studied. SDS-gel electrophoresis revealed that each agent caused intermolecular thiol crosslinking of both myosins dissociable by excess of beta-mercaptoethanol. In the simultaneous presence of H2O2 and myoglobin or H2O2 and free hemin, myosin formed covalent aggregates undissociable by beta-mercaptoethanol and therefore assessed to formation of non S-S inter molecular covalent bonds. The latter aggregates are suggested to result from pairing of myosin radicals formed by the H2O2 induced ferryl iron state in myoglobin, free hemin or hemo-myosin.     

Crystal structures of modified myoglobins. I. Heme orientation and structural changes around heme in myoglobins reconstituted with isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme

The crystal structures of sperm whale metmyoglobins reconstituted with four modified hemes, isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme, have been determined and refined at 2.2 A resolution to R = 0.217, 0.218, 0.213, and 0.222, respectively. All the crystals of these myoglobins are isomorphous with that of native metmyoglobin. The structural changes of the modified myoglobin from the native myoglobin were examined on difference Fourier maps; the orientation of 4-ethyldeuteroheme in the heme pocket is such that the heme is rotated by 180 degrees about an axis through the alpha-gamma-meso carbons, whereas the orientations of the other three hemes are the same as that of the protoheme in the native myoglobin. The changes of the structures around the heme become greater in the order of isopemptoheme, 2-ethyldeuteroheme less than pemptoheme less than 4-ethyldeuteroheme. The magnitudes of the changes seem to be related to the oxygen affinities of these four reconstituted myoglobins.