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.     

The dimer-monomer conversion of Cerithidea myoglobin coupled with the heme iron oxidation

With regard to the distal (E7) residue, gastropod sea mollusc contains both types of myoglobin, one with and the other lacking the distal histidine. We have isolated a myoglobin from the radular muscle of Cerithidea rhizophorarum, a small whelk found on the Japanese coast. Unlike Aplysia myoglobin having a single histidine residue at position 95, Cerithidea myoglobin contains three histidines at positions 48, 66 and 98. Moreover, Cerithidea MbO₂ exists as homodimers and is oxidized, not to the usual form of metMb but to the hemichrome monomers. It was also found that the hemichrome monomers thus produced can easily be converted back to the dimerized oxy-form, if the ferric protein was reduced carefully with a slight excess of sodium hydrosulfite. This dimer-monomer conversion coupled with the heme-iron oxidation in Cerithidea myoglobin is very unique, and the distal histidine at position 66 is probably responsible for its reversible formation of hemichrome from the ferric met-form that occurred transiently in due course of the oxidation reaction of Cerithidea MbO₂     

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.     

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.     

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.     

Proton NMR study of the heme environment in bacterial quinol oxidases

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.     

Dynamics of heme in hemoproteins: proton NMR study of myoglobin reconstituted with iron 3-ethyl-2-methylporphyrin

The asymmetric 3-ethyl-2-methylporphyrin iron complex was synthetized and inserted into apomyoglobin. UV-visible spectroscopic studies demonstrated the capacity of iron to coordinate different exogenous axial ligands in ferrous and ferric forms. The position of synthetic heme into the hydrophobic pocket of the reconstituted myoglobin was investigated by ((1))H NMR spectroscopy. In absence of exogenous ligand, signals of the synthetic prosthetic group were not detected, suggesting a rotational disorder of the synthetic porphyrin into the heme pocket. This direct interconversion behavior is favored since site-specific interactions between the poorly substituted heme and protein in the chiral hydrophobic cavity were weak. Complexion of cyanide to the iron allowed to quench partially the heme reorientation and two interconvertible forms, around the meso-Cα-Cγ axis, were detected in solution.     

Characterization of heme orientational disorder in myoglobin by proton nuclear Overhauser effects

Freshly reconstituted sperm whale myoglobin is a mixture of two components distinguishable by proton nuclear magnetic resonance. The two species are interconvertible and the equilibrium composition is about 90% of one form, the form studied by X-ray methods. We have used the nuclear Overhauser effect to characterize the other (minor) component in its metcyano complex. Whereas in the major form there is dipolar contact between residue 99 and the heme pyrrole ring III, in the minor form the same residue is in contact with pyrrole IV, related to ring III by a 180 degrees rotation about the alpha-gamma meso axis. This interaction proves the validity of the heme rotational disorder proposition and confirms that the apoprotein does not discriminate between the two sides of the heme in the rapid insertion process. It is proposed that the differences in nuclear Overhauser effect between the protein matrix and the heme moiety can be used to define qualitatively the structural consequences of this heterogeneity. The altered heme-protein contacts could be related to the enhanced oxygen affinity in the minor form.     

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.     

A spin label study of lipid oxidation catalyzed by heme proteins

Rapid loss of the electron spin resonance signal from a variety of spin labels is observed when ferricytochrome c or metmyogloblin are combined with lipids. Evidence is presented that this loss of signal can be used as a sensitive method to study lipid oxidation catalyzed by heme proteins. Under aerobic conditions and with lipids which bind the heme protein, the kinetics of the oxidation process as observed by the spin label method are identical to the kinetics previously observed by measurements of oxygen uptake. Use of pre-oxidized lipids under anaerobic conditions indicates that cytochrome c reacts with a product of lipid oxidation. Kinetic studies of the anaerobic reaction indicate that cytochrome c reacts rapidly with lipid oxidation products in membrane areas far larger than the area occupied by cytochrome c, implying rapid transport of reactive species within the membrane interior in directions parallel to the membrane surface. Under anaerobic conditions, reaction of cytochrome c with lipid oxidation products appears to produce a relatively long lived (hours) species located in the hydrophobic portion of the membrane, which is capable of subsequent reaction with lipid-soluble spin labels.     

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR study of molecular structure of the heme cavity

The molecular structure of the active site of myoglobin from the shark, Galeorhinus japonicus, has been studied by 1H-NMR. Some hyperfine-shifted amino acid proton resonances in the met-cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two-dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrE10 and IleFG5 residues relative to the heme in G. japonicus met-cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM (magnetic anisotropy tensor determination utilizing heme methyls) [Yamamoto, Y., Nanai, N. & Ch?j?, R. (1990) J. Chem. Soc., Chem. Commun., 1556-1557] and the results were compared with the crystal structure of P. catodon carbonmonoxy myoglobin [Hanson, J. C. & Schoenborn, B. P. (1981) J. Mol. Biol. 153, 117-124]. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.     

The electronic state of heme in cytochrome oxidase II. Oxidation-reduction potential interactions and heme iron spin state behavior observed in reductive titrations.

Magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and optical absorption spectroscopies have been used to monitor the concentrations of oxidized and reduced heme and copper during stoichiometric reductive titrations of purified beef heart cytochrome oxidase. The MCD data are deconvoluted to obtain the concentrations of reduced cytochromes a and a3 during the titrations; analysis of the EPR spectra provides complementary data on the concentrations of the EPR-detectable species. For the native enzyme in the absence of exogenous ligands, cytochromes a and a3 are reduced to approximately the same extent at all points in the titration. The reduction of the EPR-detectable copper, on the other hand, initially lags the reduction of the two cytochromes but in the final stages of the titration is completely reduced prior to either cytochrome a or a3. These non-Nernstian titration results are interpreted to indicate that the primary mode of heme-heme interaction in cytochrome oxidase involves shifts in oxidation-reduction potential for each of the two cytochromes such that a change in oxidation state for one of the hemes lowers the oxidation-reduction potential of the second heme by approximately 135 mV. In these titrations high spin species are detected which account for 0.25 spin/oxidase maximally. Evidence is presented to indicate that at least some of these signals can be attributed to cytochrome a3+ which has undergone a low-spin to high-spin state transition in the course of the titration. In the presence of carbon monoxide the oxidation-reduction properties of cytochromes a and a3 are markedly altered. The a32+. CO complex is fully formed prior to reduction of either cytochrome a3+ or the EPR-detectable copper. The g = 3 EPR signal attributed to cytochrome a3+ decreases as the MCD intensity of cytochrome a2+ increases; no significant high-spin intensity is observed at any intermediate stage of reduction. We interpret these Nernstian titration results to indicate that in the presence of ligands the oxidation-reduction potential of cytochrome a relative to cytochrome a3 is determined by the oxidation-reduction state of the stabilized cytochrome a3 ligand complex; if ligand binding occurs to reduced cytochrome a3 then cytochrome a titrates with a lower potential; cytochrome a titrates with a higher potential if oxidized cytochrome a3 is stabilized by ligand binding.     

A spin label study of lipid oxidation catalyzed by heme proteins.

Rapid loss of the electron spin resonance signal from a variety of spin labels is observed when ferricytochrome c or metmyoglobin are combined with lipids. Evidence is presented that this loss of signal can be used as a sensitive method to study lipid oxidation catalyzed by heme proteins. Under aerobic conditions and with lipids which bind the heme protein, the kinetics of the oxidation process as observed by the spin label method are identical to the kinetics previously observed by measurements of oxygen uptake. Use of pre-oxidized lipids under anaerobic conditions indicates that cytochrome c reacts with a product of lipid oxidation. Kinetic studies of the anaerobic reaction indicate that cytochrome c reacts rapidly with lipid oxidation products in membrane areas far larger than the area occupied by cytochrome c, implying rapid transport of reactive species within the membrane interior in directions parallel to the membrane surface. Under anaerobic conditions, reaction of cytochrome c with lipid oxidation products appears to produce a relatively long lived (hours) species located in the hydrophobic portion of the membrane, which is capable of subsequent reaction with lipid-soluble spin labels.     

Proton magnetic resonance study of the influence of chemical modification, mutation, quaternary state, and ligation state on dynamic stability of the heme pocket in hemoglobin as reflected in the exchange of the proximal histidyl ring labile proton

Proton nuclear magnetic resonance spectroscopy has been utilized to investigate the rates of exchange with deuterium of the proximal histidyl ring protons in a series of chemically modified and mutated forms of Hb A. Differences in rates of exchange are related to differences in the stability of the deformed or partially unfolded intermediates from which exchange with bulk solvent takes place. Each modified/mutated Hb exhibited kinetic subunit heterogeneity in the reduced ferrous state, with the alpha subunit exhibiting faster exchange than the beta subunit. Modification or mutation resulted in significant increases in the His F8 ring NH exchange rates primarily for the affected subunit and only if the modification/mutation occurs at the allosterically important alpha 1 beta 2 subunit interface. Moreover, this enhancement in exchange rate is observed primarily in that quaternary state of the modified/mutated Hb in which the modified/substituted residue makes the intersubunit contact. This confirms the importance of allosteric constraints in determining the dynamic properties of the heme pocket. Using modified or mutated Hbs that can switch between the alternate quaternary states within a given ligation state or ligate within a given quaternary state, we show that the major portion of the enhanced exchange rate in R-state oxy Hb relative to T-state deoxy Hb originates from the quaternary switch rather than from ligation. However, solely ligation effects are not negligible. The exchange rates of the His F8 ring labile protons increase dramatically upon oxidizing the iron to the ferric state, and both the subunit kinetic heterogeneity and the allosteric sensitivity to the quaternary state are essentially abolished.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Proton NMR and spin lattice relaxation study of nucleoside di- and triphosphates in neutral aqueous solutions

The average conformations of adenosine, inosine and guanosine di- and triphosphates in neutral aqueous solution have been investigated by ¹H vicinal couplings, chemical shifts and T₁ relaxation time measurements at 250 MHz. Comparison of chemical shifts with those of the corresponding nucleotide monophosphates suggests that the β-phosphate group is in all cases oriented towards the base and close to H3′. The vicinal coupling constants indicate that the proportion of the S conformer of the ribose moiety is 55–60% and that the gauche-gauche rotamer of the CH₂-OP exocyclic group is predominant.The preferential orientations of the base have been determined by minimization of the standard deviation about the mean of the molecular reorientation correlation times derived from the H8, H1′, H2′ and H3′ relaxation times and computed interproton distances. The problem of the correlation between the syn-anti equilibrium and the N S interconversion has been examined. Typical magnetization recovery curves after a 180° pulse have been simulated in the case of ATP, taking into account cross relaxation effects. It is shown that in most of the molecules under consideration the syn orientation of the base is predominant whereas for ATP the syn and anti are equivalent.     

Effects of reduction and ligation of heme iron on the thermal stability of heme-hemopexin complexes

Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (K_d < 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-hemopexin unfolds with a T_m of 52°C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (T_m 63°C). Bis-histidyl heme coordination by hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-hemopexin (T_m 55.5°C) to the ferro-heme form (T_m 48°C), and NaCl stabilizes both complexes by 10-15°C. CO binding to ferro-heme-hemopexin restores complex stability (T_m 67°C). Upon cooling, unfolded apo- and ferri-heme-hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the T_m of heme complexes by 5-12°C. The stability of the apo-N-terminal domain (T_m 53°C) is similar to that of intact hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have T_m values of 53°C, 33°C, and 75°C, respectively.     

Proton NMR and spin lattice relaxation study of nucleoside di- and triphosphates in neutral aqueous solutions

The average conformations of adenosine, inosine and guanosine di- and triphosphates in neutral aqueous solution have been investigated by 1H vicinal couplings, chemical shifts and T1 relaxation time measurements at 250 MHz. Comparison of chemical shifts with those of the corresponding nucleotide monophosphates suggests that the beta-phosphate group is in all cases oriented towards the base and close to H3'. The vicinal coupling constants indicate that the proportion of the S conformer of the ribose moiety is 55--60% and that the gauche-gauche rotamer of the CH2-OP exocyclic group is predominant. The preferential orientations of the base have been determined by minimization of the standard deviation about the mean of the molecular reorientation correlation times derived from the H8, H1', H2' and H3' relaxation times and computed interproton distances. The problem of the correlation between the syn-anti equilibrium and the N equilibrium S interconversion has been examined. Typical magnetization recovery curves after a 180 degree pulse have been simulated in the case of ATP, taking into account cross relaxation effects. It is shown that in most of the molecules under consideration the syn orientation of the base is predominant whereas for ATP the syn and anti are equivalent.