NMR studies of the conformations of leghemoglobins from soybean and lupin

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

Structural determinants in the group III truncated hemoglobin from Campylobacter jejuni

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 alpha-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-A resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III.     

Crystal structure of cytoglobin: the fourth globin type discovered in man displays heme hexa-coordination

Cytoglobin is a recently discovered hemeprotein belonging to the globin superfamily together with hemoglobin, myoglobin and neuroglobin. Although distributed in almost all human tissues, cytoglobin has not been ascribed a specific function. Human cytoglobin is composed of 190 amino acid residues. Sequence alignments show that a protein core region (about 150 residues) is structurally related to hemoglobin and myoglobin, being complemented by about 20 extra residues both on the N and C termini. In the absence of exogenous ligands (e.g. O2), the cytoglobin distal HisE7 residue is coordinated to the heme Fe atom, thus decreasing the ligand affinity. The crystal structure of human cytoglobin (2.1 A resolution, 21.3% R-factor) highlights a three-over-three alpha-helical globin fold, covering residues 18-171; the 1-17 N-terminal, and the 172-190 C-terminal residue segments are disordered in both molecules of the crystal asymmetric unit. Heme hexa-coordination is evident in one of the two cytoglobin chains, whereas alternate conformation for the heme distal region, achieving partial heme penta-coordination, is observed in the other. Human cytoglobin displays a large apolar protein matrix cavity, next to the heme, not related to the myoglobin cavities recognized as temporary ligand docking stations. The cavity, which may provide a heme ligand diffusion pathway, is connected to the external space through a narrow tunnel nestled between the globin G and H helices.     

A 1H NMR comparison of the met-cyano complexes of elephant and sperm whale myoglobin. Assignment of labile proton resonances in the heme cavity and determination of the distal glutamine orientation from relaxation data

The met-cyano complex of elephant myoglobin has been investigated by high field 1H NMR spectroscopy, with special emphasis on the use of exchangeable proton resonances in the heme cavity to obtain structural information on the distal glutamine. Analysis of the distance dependence of relaxation rates and the exchange behavior of the four hyperfine shifted labile proton resonances has led to the assignment of the proximal His-F8 ring and peptide NHs and the His-FG3 ring NH and the distal Gln-E7 amide NH. The similar hyperfine shift patterns for both the apparent heme resonances as well as the labile proton peaks of conserved resonances in elephant and sperm whale met-cyano myoglobins support very similar electronic/molecular structures for their heme cavities. The essentially identical dipolar shifts and dipolar relaxation times for the distal Gln-E7 side chain NH and the distal His-E7 ring NH in sperm whale myoglobin indicate that those labile protons occupy the same geometrical position relative to the iron and heme plane. This geometry is consistent with the distal residue hydrogen bonding to the coordinated ligand. The similar rates and identical mechanisms of exchange with bulk water of the labile protons for the three conserved residues in the elephant and sperm whale heme cavity indicate that the dynamic stability of the proximal side of the heme pocket is unaltered upon the substitution (His----Gln). The much slower exchange rate (by greater than 10(4] of the distal NH in elephant relative to sperm whale myoglobin supports the assignment of the resonance to the intrinsically less labile amide side chain.     

New transient species of sperm whale myoglobin in photodissociation of dioxygen from oxymyoglobin

We carried out the flash photolysis of oxy complexes of sperm whale myoglobin, cobalt-substituted sperm whale myoglobin, and Aplysia myoglobin. When the optical absorption spectral changes associated with the O2 rebinding were monitored on the nanosecond to millisecond time scale, we found that the transient spectra of the O2 photoproduct of sperm whale myoglobin were significantly different from the static spectra of deoxy form. This was sharply contrasted with the observations that the spectra of the CO photoproduct of sperm whale myoglobin and of the O2 photoproducts of cobalt-substituted sperm whale myoglobin and Aplysia myoglobin are identical to the corresponding spectra of their deoxy forms. These results led us to suggest the presence of a fairly stable transient species in the O2 photodissociation from the oxy complex of sperm whale myoglobin, which has a protein structure different from the deoxy form. We denoted the O2 photo-product to be Mb*. In the time-resolved resonance Raman measurements, the nu Fe-His mode of Mb* gave the same value as that of the deoxy form, indicating that the difference in the optical absorption spectra is possibly due to the structural difference at the heme distal side rather than those of the proximal side. The structure of Mb* is discussed in relation to the dynamic motion of myoglobin in the O2 entry to or exit from the heme pocket. Comparing the structural characteristics of several myoglobins employed, we suggested that the formation of Mb* relates to the following two factors: a hydrogen bonding of O2 with the distal histidine, and the movement of iron upon the ligation of O2.     

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.     

FTIR and resonance Raman studies of nitric oxide binding to H93G cavity mutants of myoglobin.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe-NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N-O vibrational frequency nuN(-O) for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that nu(N-O) shifts (by as much as 26 cm(-1)) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while nu(N-O) is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe-NO vibrational frequency nu(Fe-NO) as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that nu(Fe-NO) is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that nu(N-O) and nu(Fe-NO) are not quantitatively correlated for the MbNO complexes presented here. Furthermore, nu(N-O) and nu(Fe-NO) do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of pi-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Structural dynamics of myoglobin: spectroscopic and structural characterization of ligand docking sites in myoglobin mutant L29W

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.     

X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin at 2.0 A resolution. Stabilization of the fluoride ion by hydrogen bonding to Arg66 (E10)

The X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin has been solved and refined at 2.0 A resolution; the crystallographic R-factor is 13.6%. The fluoride ion binds to the sixth co-ordination position of the heme iron, 2.2 A from the metal. Binding of the negatively charged ligand on the distal side of the heme pocket of this myoglobin, which lacks the distal His, is associated with a network of hydrogen bonds that includes the fluoride ion, the residue Arg66 (E10), the heme propionate III, three ordered water molecules and backbone or side-chain atoms from the CD region. A comparison of fluoride and oxygen dissociation rate constants of A. limacina myoglobin, sperm whale (Physeter catodon) myoglobin and Glycera dibranchiata monomeric hemoglobin, suggests that the conformational readjustment of Arg66 (E10) in A. limacina myoglobin may represent the molecular basis for ligand stabilization, in the absence of a hydrogen-bond donor residue at the distal E7 position.     

On the Pathways of Biologically Relevant Diatomic Gases through Proteins. Dioxygen and Heme Oxygenase from the Perspective of Molecular Dynamics

This work deals with dioxygen (O₂) binding sites and pathways through inducible human heme oxygenase (HO‐1). The experimentally known distal binding site 1, and sites 2–3 above it, could be reproduced by means of non‐deterministic random‐acceleration molecular‐dynamics (RAMD) simulations. In addition, RAMD revealed the proximal binding site 5, a deeply‐seated binding site 4, which lies behind heme, as well as a few gates communicating with the external medium. In getting from site 1 to the main gate, which lies on the protein front opposed to site 4, O₂ follows chiefly the shortest direct pathway. Less frequently, O₂ visits intermediate sites 2, 4, or 5 along longer pathways. A similarity between HO‐1, myoglobin, and cytoglobin in using, for diatomic gas delivery, the direct shortest pathway from the heme center to the surrounding medium, is emphasized. Otherwise, comparing other proteins and diatomic gases, each system reveals its peculiarities as to sites, gates, and pathways. Thus, relating these properties to the physiological functions of the proteins remains in general a challenge for future studies.     

X-ray structure and refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 A resolution

The structure of carbon-monoxy (Fe II) myoglobin at 260 K has been solved at a resolution of 1.5 A by X-ray diffraction and a model refined against the X-ray data by restrained least-squares. The CO ligand is disordered and distorted from the linear conformation seen in model compounds. At least two conformations, with Fe--C--O angles of 140 degrees and 120 degrees, are required to model the system. The heme pocket is significantly larger than in deoxy-myoglobin because the distal residues have relaxed around the ligand; the largest displacement occurs for the distal histidine side-chain, which moves more than 1.4 A on ligand binding. The side-chain of Arg45 (CD3) is disordered and apparently exists in two equally populated conformations. One of these does not block the motion of the distal histidine out of the binding pocket, suggesting a mechanism for ligand entry. The heme group is planar (root-mean-square deviation from planarity is 0.08 A) with no doming of the pyrrole groups. The Fe--N epsilon 2 (His93) bond length is 2.2 A and the Fe--C bond length in the CO complex is 1.9 A. The iron is the least-squares plane of the heme, and this leads to the proximal histidine moving by 0.4 A relative to its position in deoxy-myoglobin. This shift correlates with a global structural change, with the proximal part of the molecule translated towards the heme plane.     

Tyrosine B10 inhibits stabilization of bound carbon monoxide and oxygen in soybean leghemoglobin

Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of a library of E7, E11, and B10 mutants indicate significant differences in the role of electrostatic interactions in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin, strong hydrogen bonds from several closely related conformations of the distal histidine (His(E7)) side chain preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of His(E7) is confined to a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of Tyr(B10) appears to "fix" the position of His(E7), probably by donating a hydrogen bond to the Ndelta atom of the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established by a favorable staggered geometry of the proximal histidine. The interaction between His(E7) and Tyr(B10) prevents overstabilization of bound oxygen. If hydrogen bonding from His(E7) were as strong as it is in mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport in root nodules.     

Trans-substitution of the proximal hydrogen bond in myoglobin: I. Structural consequences of hydrogen bond deletion

The structural role of a side-chain to side-chain protein hydrogen bond is examined using trans-substitution of the proximal histidine of myoglobin with methylimidazoles (Barrick, Biochemistry 1994;33:6546-6554). Modification of the chemical structure of exogenous ligands allows this hydrogen bond to be disrupted. Comparison of the crystal structures of H93G myoglobin complexed 4-methylimidazole (4meimd; methylation at carbon 4) and 1-methylimidazole (1meimd; methylation at the adjacent nitrogen, preventing hydrogen bonding between the imidazole ligand and the protein) shows that the polypeptide, heme, and methylimidazole orientations are the same within error. For 4meimd there appear to be major and minor conformations corresponding to different tautomeric states of the ligand. Conformational heterogeneity is also seen in the hyperfine-shifted region of the NMR spectrum of 4meimd complexed with high-spin H93G deoxyMb. The major conformation of the 4meimd ligand and the 1meimd ligand, as seen in the respective crystal structures, are quite similar except that the proximal ligand NH-to-Ser92-OH hydrogen bond is eliminated in the 1meimd complex, and instead the proximal ligand CH is adjacent to the Ser92-OH. Thus, this system provides a means to eliminate the Mb proximal hydrogen bond in a chemically and structurally conservative way.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.     

High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.     

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.     

Crystal structure of rat apo-heme oxygenase-1 (HO-1): mechanism of heme binding in HO-1 inferred from structural comparison of the apo and heme complex forms

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.     

1H NMR study of labile proton exchange in the heme cavity as a probe for the potential ligand entry channel in myoglobin

The exchange rates of heme cavity histidine nitrogen-bound protons in horse and dog metcyanomyoglobins have been determined at 40 degrees C as a function of pH by 1H NMR spectroscopy. They were compared to the results reported for the sperm whale homologue [Cutnell, J. D., La Mar, G. N., & Kong, S. B. (1981) J. Am. Chem. Soc. 103, 3567-3572]. The rate profiles suggest that the exchange follows EX2-type kinetics, and the relative rate values favor a penetration model over a local unfolding model. It was found that the behavior of protons located on the proximal side of the heme is similar in the three proteins. The distal histidyl imidazole NH, however, shows a highly accelerated hydroxyl ion catalyzed rate in horse and dog myoglobins relative to that in sperm whale myoglobin. NMR spectral and relaxational characteristics of the assigned heme cavity protons indicate that the global geometry of the heme pocket is highly conserved in the ground-state structure of the three proteins. We propose a model that attributes the different distal histidine exchange behavior to the relative dynamic stability of the distal heme pocket in dog or horse myoglobin vs. sperm whale myoglobin. This model involves a dynamic equilibrium between a closed heme pocket as found in metaquomyoglobin [Takano, T. (1977) J. Mol. Biol. 110, 537-568] and an open pocket as found in phenylmetmyoglobin [Ringe, D., Petsko, G. A., Kerr, D. E., & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4].(ABSTRACT TRUNCATED AT 250 WORDS)