Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.     

Studies on erythrocruorin. VII. Reconstitution of earthworm erythrocruorin from the apoprotein.

Apoerythrocruorin prepared from the giant respiratory hemoprotein of the earthworm (60 S, Mr = 3 X 10(-6)) is an electrophoretically homogeneous molecule which sediments as a single peak of low molecular weight (3.5 S) and has a lower alpha-helical content (approx. 30%) than the native protein. Titration of globin with ferric heme indicates the presence of different binding sites; however, after purification by ion exchange chromatography, the reconstitution product contains 1 haem/23 000 g of protein as the native molecule. Reconstituted ferric erythrocruorin is a low molecular weight hemichrome with the same optical and physicochemical properties of the hemichrome formed by natural ferric erythrocruorin. Reconstituted ferrous erythrocruorin reacquires the alpha-helical content and the quaternary structure of the native molecule. Reassociation into 10-S speices (1/12 of the whole molecules) is fast and easy, while that into whole molecules is slow and somewhat erratic. The functional properties of reconstituted ferrous erythrocruorin (oxygen affinity, cooperativity in oxygen binding, magnitude of Bohr effect) are very similar to those of the "stable" low cooperativity form of the undissociated protein.     

Ligand binding properties of horse hemoglobins containing deutero- and mesoheme.

The reactions of horse globin reconstituted with proto-, deutero-, and mesoheme have been examined by equilibrium and kinetic methods. In virtually all reactions studied, mesohemoglobin displays the more extreme functional behavior, whereas deuterohemoglobin exhibits behavior which is either very similar to native hemoglobin or intermediate between the two. Our kinetic and equilibrium results indicate that the primary effect of heme modification on the functional properties of hemoglobin is to alter the intrinsic reactivities of the deoxy and liganded conformations. Heme modification does not, however, result in substantial alterations in the conformational equilibrium between the two states. Simple inductive electronic effects of the 2- and 4-substituents of the heme moiety in deutero- and mesohemoglobin are apparently not sufficient to explain the observed equilibrium and kinetic properties completely, which indicates that steric effects of these substituents may also play a role in determining the functional behavior of the hemoglobin molecule.     

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.     

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.     

Tension at the heme: a mathematical treatment of hemoglobin cooperativity

The unique feature of this model is that both the fractional saturation and the free energy change are handled within the framework of the tension-displacement mechanism for hemoglobin co-operativity proposed by Perutz (1970, 1972), i.e. heme iron movement and associated changes in the protein globin internal tension, tau. Physically, tau is the force applied by the protein globin on the proximal histidine, preventing the iron stereochemistry from attaining the geometry preferred in the bound state. It is assumed that a change in position of the heme iron on ligand binding displaces the protein globin proportionately, thereby decreasing tau at neighboring sites; the resulting energy change is assumed to be delocalized throughout the flexible protein globin rather than localized at the heme group per se. The physical interpretation of the model parameters has important implications with regard to data analysis: first, structural data is used to fix the molecular displacements lt and lr; second, jt/jr provides a measure of the protein's intrinsic (i.e. tau = 0) affinity for the bound ligand, and third the set [Ei] is a property of the hemoglobin molecule only and can be determined, in principle, using structural data and optical absorption spectra. The calculated protein globin internal tension in the tense, unbound state (approximately 2 X 10(-5) dyne), determined from the fractional saturation data of Joels & Pugh (1958), is very similar (approximately 3.2 X 10(-5) dyne) to the value estimated by Hopfield (1973) from free energy considerations.     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.Abbreviations used GMHX the holo-protein (including b-type heme, Glycera dibranchiata monomer hemoglobin Component X (X=2, 3, or 4) - GMGX the apo-protein, or globin, Glycera dibranchiata monomer globin derived from Component X (X=2, 3, or 4) - rec-gmg the globin derived from a recombinant holoprotein of a Glycera dibranchiata monomer hemoglobin, rec-gmh, whose sequence has been inferred from an isolated cDNA insert - CB label refers to peptides generated from cyanogen bromide cleavage of GMG4 - HPLC high-performance liquid chromatography - T label refers to peptides generated from trypsin digests of GMG4 - Mb myoglobin - MCS monomer hemoglobin crystal structure from Glycera dibranchiata. H, N-terminal sequence of GMG4 - SWMb sperm whale myoglobin     

The structure of metmanganoglobin

The structure of metmanganoglobin, in which Mn(III) replaces Fe(III) as the heme metal, has been compared with that of native hemoglobin by X-ray difference Fourier techniques. Their quaternary structures are identical and their tertiary structures are similar, as expected both from the close stereochemical correspondence between manganese porphyrins and the corresponding iron porphyrins, and from the similarities in functional properties previously reported. There are, however, a number of small but significant differences. The α-heme is essentially unperturbed by the metal substitution, in contrast to the β-heme. It appears that the sixth ligand of the β-heme, a water molecule in native hemoglobin, has been lost, and that the resultant five-co-ordinate β-heme in metmanganoglobin is distinctly ruffled in accord with quasi-S₄ symmetry. The loss of non-covalent ligand-globin interactions together with the heme ruffling produce numerous small perturbations in the β-globin, which are transmitted across the α₁-β₁ interface to the α-globin. The α₁-β₂ interface is only slightly perturbed. Metmanganoglobin thus displays some, but not all, of the structural features to be expected in a partially liganded hemoglobin: those arising directly from ligand loss from the β-hemes, and contraction of the ligand pocket in the β-chain, but not others, such as those which accompany a marked alteration in the distance of the proximal histidine from the mean plane of the porphyrin. The basic similarity in the structural and functional properties of manganoglobin and native hemoglobin demonstrates that hemoglobin function is not uniquely dependent on the co-ordination properties of the metal.     

Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins

Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly effected by the coordination and noncovalent chemical environment imposed by its connate protein. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here we explore this question by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria, including human neuroglobin and cytoglobin, a nonsymbiotic hemoglobin from rice, and a truncated hemoglobin from the cyanobacterium Synechocystis. This is done with a set of experiments measuring the reduction potentials of each wild-type hxHb and its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a noncoordinating side chain. These reduction potentials, coupled with studies of the mutant proteins saturated with exogenous imidazole, enable us to assess the effects of the protein matrices on histidine coordination. Our results show significant variation among the hxHbs, demonstrating flexibility in the globin moiety's ability to regulate reversible coordination. This regulation is particularly evident in the plant nonsymbiotic hemoglobins, where ferric state histidine coordination affinity is substantially lowered by the protein matrix.     

A possible new control mechanism suggested by resonance Raman spectra from a deep ocean fish hemoglobin

The rattail fish, Coryphaenoides armatus, lives at ocean depths of 3000 m. As an adaptation for pumping oxygen into the swim bladder against the extreme pressures at the ocean bottom, the hemoglobin from this fish at low pH exhibits an extraordinarily low affinity for ligands. In this study, continuous wave and time-resolved Raman techniques are used to probe the binding site in this hemoglobin. The findings show an association between the low-affinity material and a highly strained heme-proximal histidine linkage. The transient Raman studies reveal differences in the protein structural dynamics at pH 6 and 8. The emerging picture derived from both this and earlier studies is that in vertebrate hemoglobins the heme-proximal histidine linkage represents a key channel through which species- and solution-dependent variations in the globin are communicated both statically and dynamically to the heme to produce an extensive range of ligand binding properties. Also presented is a new model that relates both intensity and frequency of the resonance Raman band involving the iron-proximal histidine stretching mode to specific protein controlled structural degrees of freedom. There emerges from this model a mechanism whereby modifications in the proximal heme pocket can further reduce the affinity of an already highly strained T state structure of hemoglobin.     

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.     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.     

Glycera dibranchiata hemoglobin. Structure and refinement at 1.5 A resolution

The coelomic cells of the common marine bloodworm Glycera dibranchiata contain several hemoglobin monomers and polydisperse polymers. We present the refined structure of one of the Glycera monomers at 1.5 A resolution. The molecular model for protein and ordered solvent for the deoxy form of the Glycera monomer has been refined to a crystallographic R-factor of 12.7% against an X-ray diffraction dataset at 1.5 A resolution. The positions of 1095 protein atoms have been determined with a maximum root-mean-square (r.m.s.) error of 0.13 A, and the r.m.s. deviation from ideal bond lengths is 0.015 A and from ideal bond angles is 1.0 degree. The r.m.s. deviation of planar groups from their least-squares planes is 0.007 A, and the r.m.s. deviation for torsion angles is 1.2 degrees for peptide groups and 16.8 degrees for side-chains. A total of 153 water molecules has been located, and they have been refined to a final average occupancy of 0.80. Multiple conformations have been found for five side-chains, and a change has been suggested for the sequence at five residues. The heme group is present in the "reverse" orientation that differs only in the positions of the vinyl beta-carbons from the "normal" orientation. The doming of the heme towards the proximal side, and the bond distances and angles of the heme and proximal histidine are typical of most deoxy globin structures. The substitution of leucine for the distal histidine residue (E7) creates an unusually hydrophobic heme pocket.     

Nitric oxide can modify amino acid residues in proteins.

Nitric oxide derived from sodium nitroprusside binds to the heme moiety of hemoglobin and also modifies some functional groups in the protein. As hemoglobin concentration is increased, globin modification is decreased presumably due to formation of the NO complex with heme. The SH groups of hemoglobin are probably not involved in the formation of the stable product formed by NO. In the presence of inositol hexaphosphate, which binds preferentially in the cleft between the two beta-chains of hemoglobin, formation of one modified derivative was selectively reduced. With hemoglobin specifically blocked on its N-terminal residues, globin modification was also significantly reduced. Carbonic anhydrase, which is blocked at its N-terminus, was also refractory to modification. The results suggest that the N-terminal groups of some proteins can be modified by nitric oxide, perhaps by deamination.     

The nerve hemoglobin of the bivalve mollusc Spisula solidissima: molecular cloning, ligand binding studies, and phylogenetic analysis

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P(50) approximately 0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.     

Characterization by NMR of the heme-myoglobin adduct formed during the reductive metabolism of BrCCl3. Covalent bonding of the proximal histidine to the ring I vinyl group

The reductive debromination of BrCCl3 by ferrous deoxymyoglobin leads to the covalent bonding of the prosthetic heme to the protein. We have previously shown, by the use of peptide mapping and mass spectrometry, that histidine residue 93 is covalently bound to the heme moiety. In the present study the structure of the heme adduct was more completely determined by 1H and 13C NMR techniques. We have found that the ring I vinyl group of the prosthetic heme was altered by the addition of a histidine imidazole nitrogen to the alpha-carbon and a CCl2 moiety to the beta-carbon. The electronic absorption spectra of the oxidized and reduced states of the altered heme-protein indicated that the heme-iron exists in a bis-histidine-ligated form. Analysis of the crystal structure of native myoglobin suggested that for the altered heme-protein, histidine residues 97 and 64 are ligated to the heme-iron and that residue 97 has replaced the native proximal histidine residue 93. These movements, in effect a "histidine shuffle" at the active site, may be responsible for the enhanced reducing activity of the altered protein.     

Characterization of the heme–histidine cross-link in cyanobacterial hemoglobins from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. PCC 6803 and<Emphasis Type="Italic"> Synechococcus</Emphasis> sp. PCC 7002

The recombinant product of the hemoglobin gene of the cyanobacterium Synechocystis sp. PCC 6803 forms spontaneously a covalent bond linking one of the heme vinyl groups to a histidine located in the C-terminal helix (His117, or H16). The present report describes the ¹H, ¹⁵N, and ¹³C NMR spectroscopy experiments demonstrating that the recombinant hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002, a protein sharing 59% identity with Synechocystis hemoglobin, undergoes the same facile heme adduct formation. The observation that the extraordinary linkage is not unique to Synechocystis hemoglobin suggests that it constitutes a noteworthy feature of hemoglobin in non-N₂-fixing cyanobacteria, along with the previously documented bis-histidine coordination of the heme iron. A qualitative analysis of the hyperfine chemical shifts of the ferric proteins indicated that the cross-link had modest repercussions on axial histidine ligation and heme electronic structure. In Synechocystis hemoglobin, the unreacted His117 imidazole had a normal pK _a whereas the protonation of the modified residue took place at lower pH. Optical experiments revealed that the cross-link stabilized the protein with respect to thermal and acid denaturation. Replacement of His117 with an alanine yielded a species inert to adduct formation, but inspection of the heme chemical shifts and ligand binding properties of the variant identified position 117 as important in seating the cofactor in its site and modifying the dynamic properties of the protein. A role for bis-histidine coordination and covalent adduct formation in heme retention is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - GlbN cyanoglobin - Hb hemoglobin - hx hexacoordinate - MALDI matrix-assisted laser desorption ionization - NOE nuclear Overhauser effect - NOESY two-dimensional nuclear Overhauser effect spectroscopy - rHb recombinant hemoglobin - rHb-A recombinant hemoglobin with covalently attached heme - rHb-R recombinant heme-reconstituted hemoglobin - S6803 Synechocystis sp. PCC 6803 - S7002 Synechococcus sp. PCC 7002 - TOCSY totally correlated two-dimensional spectroscopy - TPPI time-proportional phase incrementation - trHb truncated hemoglobin - WATERGATE water suppression by gradient-tailored excitation - WEFT water elimination Fourier transform     

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 1. Effects on structure

Replacement of the axial histidine ligand with exogenous imidazole has been accomplished in a number of heme protein mutants, where it often serves to complement the functional properties of the protein. In this paper, we describe the effects of pH and buffer ion on the crystal structure of the H175G mutant of cytochrome c peroxidase, in which the histidine tether between the heme and the protein backbone is replaced by bound imidazole. The structures show that imidazole can occupy the proximal H175G cavity under a number of experimental conditions, but that the details of the interaction with the protein and the coordination to the heme are markedly dependent on conditions. Replacement of the tethered histidine ligand with imidazole permits the heme to shift slightly in its pocket, allowing it to adopt either a planar or distally domed conformation. H175G crystallized from both high phosphate and imidazole concentrations exists as a novel, 5-coordinate phosphate bound state, in which the proximal imidazole is dissociated and the distal phosphate is coordinated to the iron. To accommodate this bound phosphate, the side chains of His-52 and Asn-82 alter their positions and a significant conformational change in the surrounding protein backbone occurs. In the absence of phosphate, imidazole binds to the proximal H175G cavity in a pH-dependent fashion. At pH 7, imidazole is directly coordinated to the heme (d(Fe--Im) = 2.0 A) with a nearby distal water (d(Fe--HOH) = 2.4 A). This is similar to the structure of WT CCP except that the iron lies closer in the heme plane, and the hydrogen bond between imidazole and Asp-235 (d(Im--Asp) = 3.1 A) is longer than for WT CCP (d(His--Asp) = 2.9 A). As the pH is dropped to 5, imidazole dissociates from the heme (d(Fe--Im) = 2.9 A), but remains in the proximal cavity where it is strongly hydrogen bonded to Asp-235 (d(Im--Asp) = 2.8 A). In addition, the heme is significantly domed toward the distal pocket where it may coordinate a water molecule. Finally, the structure of H175G/Im, pH 6, at low temperature (100 K) is very similar to that at room temperature, except that the water above the distal heme face is not present. This study concludes that steric restrictions imposed by the covalently tethered histidine restrain the heme and its ligand coordination from distortions that would arise in the absence of the restricted tether. Coupled with the functional and spectroscopic properties described in the following paper in this issue, these structures help to illustrate how the delicate and critical interactions between protein, ligand, and metal modulate the function of heme enzymes.     

Deficient heme synthesis as the cause of noninducibility of hemoglobin synthesis in a Friend erythroleukemia cell line.

Friend cells of the line Fw are not induced to accumulate substantial amounts of hemoglobin and to become benzidine-positive when treated with butyric acid or other inducers, except in the presence of exogenous hemin. The cells are shown to have a deficiency in heme synthesis since they require exogenous hemin during the period of maximal hemoglobin synthesis; since endogenous heme synthesis cannot be induced to the level found in normal inducible Friend cells, even after hemoglobin synthesis has been induced by hemin and butyric acid and the hemin has then been withdrawn; since they are not inducible for ferrochelatase (heme synthetase) activity; and since they accumulate free globin chains after stimulation with butyric acid in the absence of hemlin. Comparison of globin synthesis and globin mRNA content of the cells shows that globin synthesis is not controlled by the hemin-controlled repressor of protein synthesis (HCR) nor by any specific translational control of globin synthesis by hemlin.     

Reaction of nitric oxide with heme proteins and model compounds of hemoglobin

Rates for the reaction of nitric oxide with several ferric heme proteins and model compounds have been measured. The NO combination rates are markedly affected by the presence or absence of distal histidine. Elephant myoglobin in which the E7 distal histidine has been replaced by glutamine reacts with NO 500-1000 times faster than do the native hemoglobins or myoglobins. By contrast, there is no difference in the CO combination rate constants of sperm whale and elephant myoglobins. Studies on ferric model compounds for the R and T states of hemoglobin indicate that their NO combination rate constants are similar to those observed for the combination of CO with the corresponding ferro derivatives. The last observation suggests that the presence of an axial water molecule at the ligand binding site of ferric hemoglobin A prevents it from exhibiting significant cooperativity in its reactions with NO.