Myoglobin diffusion in bovine heart muscle.

The rotational mobility of myoglobin in situ was determined by proton nuclear magnetic resonance line width measurements of a characteristic myoglobin resonance observed in bovine heart muscle spectra. The protein diffuses intracellularly at nearly half the rate observed in dilute solution. This high mobility allows the oxygenated form of myoglobin to contribute significantly to the overall diffusive flux of oxygen in respiring heart muscle.

Proton NMR investigation of the influence of subunit assembly on the low-spin in equilibrium high-spin equilibrium of met-azido hemoglobin A.

The 1H nuclear magnetic resonance signals for the side-chain labile protons of the proximal His-F8 in met-azido derivatives of the isolated chains and intact tetramer of hemoglobin have been identified. Assignment of the two peaks to the individual subunits of the intact tetramer was effected by the basis of the strong similarity of shift of one of the two peaks to that of met-azido semi-hemoglobin, where hemes occupy primarily the alpha subunits, with the heme cavity vacant in the adjacent beta subunits. The magnitudes of the hyperfine shift for both the His-F8 ring NH and the heme methyls reflect the degree of high-spin character in the thermal spin equilibrium between the high-spin, S = 5/2, and low-spin, S = 1/2, states. The changes in these shifts upon tetramer assembly demonstrate that formation of the intersubunit contacts in the R-state met-azido hemoglobin from the isolated chains causes a slight decrease in high-spin character of the alpha (22 to 20%) and a marked increase (5 to 11%) in the high-spin character of the beta subunits. The changes in spin-character are interpreted in terms of slight increase and decrease in the strength of the iron-His F8 bond upon tetramer assembly in the alpha and beta subunits, respectively. These changes in axial bonding upon forming R-state intersubunit contacts are consistent with previous observation on forming the R-state deoxy Hb tetramer from the isolated chains (Nagai, K., La Mar, G.N., Jue, T. and Bunn, H.F. (1982) Biochemistry 21, 842-847).

Proton NMR study of the thermodynamics and kinetics of the acid in equilibrium base transitions in reconstituted metmyoglobins.

Optical and proton NMR pH titrations of sperm whale metmyoglobin (metMb) in its native form and reconstituted with chemically modified hemes reveal that the pKa for the acid in equilibrium base transition decreases as the heme 2,4-substituents are made more electron withdrawing. The proton NMR spectra yields resonances which are averaged over the acidic and basic forms of the protein, but still exhibit significant exchange line broadening. Analysis of this exchange contribution to the linewidth is consistent with the simple kinetic scheme metMb+H2O + OH- k2 in equilibrium k1 metMbOH + H2O with k2 = 1.3 +/- 0.5 . 10(10) M-1 . s-1 and k1 = 1.6 +/- 0.6 . 10(5) s-1 for the native protein. The effect of electron-withdrawing substituents on the heme increase k2 and decrease k1.

Proton NMR study of model substrate binding in hemoproteins. Intercalation of mercuric triiodide in sperm whale met-aquo myoglobin.

1H-NMR spectra have been recorded for sperm whale met-aquo myoglobin intercalated with xenon, cyclopropane, mercuric triiodide and auric triiodide. All four agents are known to intercalate on the proximal side of the heme over pyrrole A. The complexes of xenon and cyclopropane exhibit insignificant shifts for all four heme methyls, suggesting that these species fit into an existing hole without causing significant perturbations on the heme cavity. Mercuric and auric triiodide, on the other hand, induce substantial changes in the hyperfine-shifts for the heme methyls. Based on the previously assigned methyl peaks in met-aquo myoglobin, we find that methyl-1, closest to the intercalating agent, is affected most, with the influence decreasing with distance from the binding site. These results indicate that determination of the relative perturbations on the assigned heme methyl shifts due to substrate binding can be utilized to ascertain the substrate-heme stereochemistry in high-spin ferric hemoproteins such as peroxidases.

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.

A nuclear overhauser study of heme orientational isomerism in monomeric Chironomus hemoglobins.

The nuclear Overhauser effect (NOE) was used to investigate heme orientation and to obtain assignments for all resolved resonances in the 1H-NMR spectrum of met-cyano Chironomus thummi thummi monomeric hemoglobins III and IV (Hb III and Hb IV). The only non-heme resolved resonance was found to be from Phe-38 (CD1), and NOE dipolar connectivity between this resonance and the heme 5- and 8-methyls was used to establish the absolute orientation of the heme for each heme-insertion isomer present. The assignments of resonances and heme disorder permitted structural comparisons between the various components, including those due to a point mutation in Hb III. Finally, the characteristic differences of NOE patterns to amino-acid protons from substituents on heme pyrroles I and II formed the basis for assigning resonances and heme orientation relative to native Hb IV for deuterohemin-reconstituted Hb IV, for which there are no X-ray data available.

Hydrogen isotope effects on the proton nuclear magnetic resonance spectrum of bovine ferricytochrome b5: axial hydrogen bonding involving the axial His-39 imidazole ligand.

The potential role of hydrogen bonding interactions in modulating the molecular and electronic structure of the active site of solubilized bovine ferricytochrome b5 has been investigated by monitoring solvent isotope effects on proton-NMR spectral parameters. It is observed that the hyperfine shifts of both the heme prosthetic group and one coordinated His are sensitive, while those for the other axial His and non-coordinated residues are insensitive, to 2H for 1H exchange. Two types of isotope influences are characterized; one whose chemical shift influence is time-resolved on the NMR time scale, and involves a single proton on one axial ligand, and a second effect which involves multiple protons, is not time resolved, and influences primarily the heme. A large isotope effect on the hyperfine shift is identified for the C beta H signals of His-39 but not His-63. The exchangeable ring NH of His-39 is assigned, and the pH influence on the exchange properties of heme pocket labile protons, when compared to the rate of base catalyzed averaging of the His-39 C beta H isotope effect, lead to the conclusion that the axial hydrogen bond which is responsible for this isotope effect is that between His-39 ring NH and Gly-42 carbonyl. The more rapid exchange of labile protons with solvent for His-63 than His-39 confirms a less solvent accessible and stronger hydrogen bonded His-39 than His-63. The stronger His-39-Gly-42 than His-63-Phe-58 hydrogen bond involving the ring NH leads to more extensive His-39 imidazolate character and hence a stronger iron-His-39 than iron-His-63 bond. The much larger hyperfine shifts for His-39 than His-63 imidazole ring non-labile protons support the stronger bonding of the former ligand, and account for the orientation of the rhombic magnetic axes by His-39 rather than His-63. The solvent isotope effect on the heme leads to rotation of the prosthetic group about the His-Fe-His bond by approximately 0.5 degrees so as to shorten the 7-propionate link to Ser-64. This suggests that the hydrogen bonds between the 7-propionate group and Ser-64 are responsible for the effect.

Proton NMR study of coordinated imidazoles in low-spin ferric heme complexes. Assignment of single proton histidine resonance in hemoproteins.

The proton signals for the coordinated axial imidazoles in a series of low-spin ferric bis-imidazole complexes with natural porphyrin derivatives have been located and assigned. The methyl signals of several methyl-substituted imidazoles have also been resolved for the mixed ligand complexes of imidazole and cyanide ion. The imidazole spectra for the bis complexes are essentially the same as those reported earlier for synthetic porphyrins, with the hyperfine shifts exhibiting comparable contributions from the dipolar and contact interactions. The contact contribution reflects spin transfer into a vacant imidazole pi orbital. The spectra of both the mono- and bis-imidazole complex concur in predicting that only the 2-H and 5-CH2 signals of an axial histidine are likely to resonate clearly outside the diamagnetic 0 to --10 ppm from TMS region in hemoproteins. However, both the 2-H and 4-H imidazole peaks are found to be too broad to detect in a hemoprotein. Hence, it is suggested that the pair of non-heme, single-proton resonances in low-spin met-myoglobin cyanides arise from the non-equivalent methylene protons at the 5-position of the histidyl imidazole. Both the resonance positions and relative linewidths in the model compounds are consistent with the data for this pair of protons in myoglobins. The possible interpretations of the average downfield bias of these signals as well as the magnitude of their spacing, are discussed in terms of the conformation of the proximal histidine relative to the heme group.

Axial pertubations on the electronic and magnetic properties of ferric porphyrins. II. Solvent effects on the proton NMR spectra of low-spin cyano complexes.

The proton NMR spectra of a series of low-spin bis-cyano ferric complexes of tetraarylporphyrins and octaethylporphyrin in a variety of solvents have been recorded and analyzed. The hyperfine shifts are shown to be very sensitive to the solvent, experiencing an overall downfield bias as the solvent hydrogen-bonding donor strength increased. The characteristic pattern of the contact and dipolar shifts for the meso-aryl group in tetraarylporphyrin complexes are shown to permit a quantitative separation of the dipolar and contact contributions to the hyperfine shift. The separated components indicate that increased solvent hydrogen bonding strength significantly decreases the magnetic anisotropy of the iron and diminishes porphyrin replaced by iron pi bonding. The changes in anisotropy with solvent are shown to be consistent with the coordinated cyanide acting as a proton acceptor. Although similar solvent effects are found to be absent in bis-imidazole complexes, a downfield bias of half the magnitude of the bis-cyano complexes is observed in mixed cyano/imidazole complexes. Hence, the heme hyperfine shifts in cyano-metmyoglobins and -hemoglobins may serve as probes for the protonation of the distal histidyl imidazole.

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.

Heme methyl hyperfine-shifted nuclear magnetic resonance peaks assigned by selective deuteration as indicators of heme-protein interactions in metmyoglobins.

The heme methyl resonances in a variety of high-spin, low-spin and spin-equilibrium forms of sperm whale metmyoglobin have been assigned by reconstituting myoglobin with selectively deuterated hemes. Two patterns for the heme methyl hyperfine shifts are observed, one characteristic of the low-spin state and the other typical of the high-spin state. The two protein forms which can change the position of their spin equilibrium significantly with changing temperature exhibit the pattern of the dominant spin state component at any temperature. The different hyperfine shift patterns for the low-spin and high-spin states are concluded to arise not from different heme-protein contacts in the two spin states, but from characteristic differential sensitivities of the dominant spin transfer mechanisms to the same rhombic perturbation.

One- and two-dimensional nuclear Overhauser effect studies of the electronic/molecular structure of the heme cavity of ferricytochrome b5.

A nuclear Overhauser effect, NOE, study of solubilized native bovine ferricytochrome b5 has provided the complete assignment of the heme resonances as well as those of the majority of the amino acid side-chains making contact with the prosthetic group. The resonances which could not be identified are those from positions very close to the iron (less than 5 A) for which paramagnetic relaxation is sufficiently strong to significantly decrease the NOEs. The observed 1H-1H dipolar contacts generally confirm a solution structure unchanged from that described in single crystals, except for the detailed orientation of the heme side-chains. The 2-vinyl group is found in both the cis and trans in-plane orientation as opposed to exclusively cis in the crystal, and the 7-propionate group is rotated by 30 degrees in solution towards the 6-propionate group. Identification of resonances for the individual axial histidine residues indicates non-equivalent interaction with the heme iron, and the patterns of meso-H, pyrrole substituent and amino acid dipolar shifts allow the location of the principal magnetic axes in the protein coordinate system. This identifies His-39 as the dominant influence in determining the electronic ground state that orients the molecular orbital for facile electron transfer via the exposed heme edge. The complete two-dimensional NOESY map for ferricytochrome b5 is presented that yields all the cross peaks expected on the basis of the one-dimensional NOE studies, and indicates that such two-dimensional methods should have profitable extension to strongly hyperfine-shifted resonances in paramagnetic proteins.

13C-NMR study of labeled vinyl groups in paramagnetic myoglobin derivatives.

The 13C-NMR spectra of high-spin met-aquo myoglobin, spin-equilibrium met-azido myoglobin, low-spin met-cyano myoglobin, deoxy myoglobin and carbonmonoxy myoglobin from sperm whale reconstituted with hemin 13C enriched at both vinyl alpha or beta positions have been recorded. In all cases the labeled vinyl 13C signals are clearly resolved and useful spectra could be obtained within approx. 15 minutes. The decoupling of multiplet structure due to attached proton(s) has led to the specific assignment of vinyl 13C alpha signals in all paramagnetic derivatives and the 13C beta signals in met-cyano myoglobin. In all other cases, the collapse of the proton multiplet structure as a function of 1H decoupling frequency has located, but not assigned, the attached 1H resonance positions which are obscured by the intense diamagnetic envelope in the 1H-NMR spectrum. The resulting vinyl 13C hyperfine shifts follow Curie behavior, and the patterns closely resemble those in the appropriate model complexes in the same oxidation/spin/ligation state, except that the protein exhibits more in-plane asymmetry. The hyperfine shift patterns are indicative of dominant pi contact shifts for all ferric complexes. Deoxy myoglobin vinyl 13C and 1H contact shifts provide little evidence for pi bonding.

Proton-NMR investigation of the heme cavity in the cyanomet derivative of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis.

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibility tensor, and the major axis found tilted from the normal in a manner similar to that found for the Fe-CO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.

High-field 1H NMR studies of synthetic analogs of somatostatin. Structural features involving aromatic residues in an active eight-membered ring analog.

360 MHz 1H-NMR data are presented for somatostatin and an analog whose primary structure is cyclo(-Gaba-Asn5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-). This report focuses on the aromatic portion of the spectrum, and this region for the analog is unambiguously assigned, using two experimental approaches: selective deuteration and photo-induced CIDNP. The most prominent feature of the analog aromatic spectrum is a two-proton resonance which exhibits a pronounced upfield shift. Significantly, this feature is also present for somatostatin and other active analogs (unpublished data). Assignments show that this resonance derives from the ortho hydrogens of the Phe6 and that aromatic resonances of Phe6 shift markedly upfield as temperature is decreased. In contrast, the aromatic resonances of Phe7,11 and DTrp8 reveal generally much smaller temperature coefficients and shift primarily downfield as temperature is decreased. Ring-current analysis shows that simple pair-wise parallel pi-stacking alone cannot give rise to the observed data. However, a simple hypothesis involving only two phenylalanine residues is totally consistent with the data if they maintain a time-averaged co-perpendicular orientation. Indirect evidence is offered which implicates only one phenylalanine stacking partner for Phe6, which we tentatively identify as Phe11.

Axial histidyl imidazole non-exchangeable proton resonances as indicators of imidazole hydrogen bonding in ferric cyanide complexes of heme peroxidases.

Proton NMR spectra of a model of low-spin cyanide complexes of ferric hemoproteins indicate that two broad single-protein resonances from the axial imidazole can be resolved outside the diamagnetic spectral region. Upon deprotonation of the imidazole in the model, the upfield resonance shifts dramatically to higher field, suggesting that its position may reflect the degree of hydrogen bonding or proton donation of the imidazole. Met-cyano myoglobin reveals a pair of such broad peaks in the regions expected for an essentially neutral axial imidazole. In the cyano complexes of horseradish peroxidase and cytochrome c peroxidase, a pair of single-proton resonances are located which are assigned to the same imidazole protons on the basis of their linewidth and shift changes upon altering the heme substituents. The upfiled proton, however, is found at much higher field than in metMbCN. The upfield bias of this resonance is taken as evidence for appreciable imidazolate character for the axial ligand in these heme peroxidases.

Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study.

The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 residue of the apoprotein. Moreover, they provide new information on the correlations existing between the shape and the intensity of the dichroic bands and the heme orientation inside the reconstituted protein. Our data suggest that positive Soret CD bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'correct' orientation (as in horse heart myoglobin), and negative dichroic bands are associated with hemoglobins possessing, at equilibrium, the heme in the so-called 'reversed' orientation (as for example, in Glycera dibranchiata hemoglobins). Thus, negligible contribution to the CD signal in reconstituted proteins can be associated to a 'wrong' orientation of the heme group, no matter whether the orientation at the equilibrium is the 'correct' or the 'reversed' one. Finally, the results obtained indicate that the perturbations due to the heme re-orientation appear as a local phenomenon, not affecting the distant domains of the macromolecule.

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.

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.

Interpretation of hyperfine shift patterns in ferricytochromes b5 in terms of angular position of the heme: a sensitive probe for peripheral heme protein interactions.

The 1H-NMR hyperfine shift pattern of the heme in a variety of low-spin ferricytochromes b5 has been analyzed in terms of the angular position of the prosthetic group within a structurally and magnetically-conserved protein matrix. A simple model is presented in which the changes in the spread of the predominantly contact shifted methyl and predominantly dipolar shifted meso-H signals of the heme, as well as shift trends for individual signals, provide sensitive indicators of the orientation of the heme relative to the orbital hole (singly-occupied d orbital), which in turn is related to the rhombic magnetic axes. The invariance of the axial His and non-coordinated residue hyperfine shifts show that it is the heme within a relatively rigid protein matrix, rather than the magnetic coordinate system, which is displaced angularly about the heme normal in order to accommodate variations in the polypeptide, orientation of the heme about the alpha,gamma-meso axis, and the length of heme carboxylate chains. Native heme shows increased counterclockwise rotation about the heme normal in the order rat-->beef-->chicken ferricytochrome b5, which is attributed largely to increased bulk of a variable sequence hydrophobic cluster consisting of residues 23, 25 and 32. The two alternate heme orientations about the alpha,gamma-meso axis are shown to also differ by rotation about the heme normal. A semiquantitative estimate of the degree of angular accommodation based on the spread of the meso-H rhombic dipolar shifts indicate rotations of 2-10 degrees. Possible functional consequences of such angular accommodation in relation to the role of these proteins in electron transfer are discussed.