Effects of mutations on the molecular dynamics of oxygen escape from the dimeric hemoglobin of Scapharca inaequivalvis (original) (raw)
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Biochemistry, 2001
Cooperative ligand binding in the dimeric hemoglobin from the blood clam Scapharca inaequiValVis results primarily from tertiary, rather than quaternary, structural changes. Ligand binding is coupled with conformational changes of key residues, including Phe 97, which is extruded from the proximal heme pocket, and the heme group, which moves deeper into the heme pocket. We have tested the role of the heme movement in cooperative function by mutating Ile 114, at the base of the heme pocket. Replacement of this residue with a Met did not disturb the hemoglobin structure or significantly alter equilibrium ligand binding properties. In contrast, substitution with a Phe at position 114 inhibits the ligand-linked movement of the heme group, and substantially reduces oxygen affinity and cooperativity. As the extent of heme movement to the normal position of the ligated state is diminished, Phe 97 is inhibited from its movement into the interface upon ligand binding. These results indicate a tight coupling between these two key cooperative transitions and suggest that the heme movement may be an obligatory trigger for expulsion of Phe 97 from the heme pocket.
Journal of Molecular Biology, 1998
A cluster of interface ordered water molecules has been proposed to act as a key mediator of intersubunit communication in the homodimeric hemoglobin of Scapharca inaequivalvis. Mutations of Thr72 to Val and Ile, which lack the hydroxyl group to hydrogen bond the deoxy interface water molecules, result in sharply altered functional properties. We have determined the high resolution (1.6 ± 1.8 A Ê) crystal structures of these two mutants in both the deoxygenated and CO-liganded states. These structures show minimal protein structural changes relative to the same native derivatives, despite greater than 40-fold increases in oxygen af®nity. In the deoxy state of both mutants two water molecules at the periphery of the water cluster are lost, and the remaining cluster water molecules are destabilized. The CO-liganded structures show key differences between the two mutants including a more optimal interface packing involving Ile72 that acts to stabilize its high af®nity (R) state. This additional stabilization allows rationalization of its lowered cooperativity within the context of a two-state model. These studies support a key role of ordered water in cooperative functioning and illustrate how subtle structural alterations can result in signi®cantly altered functional properties in an allosteric molecule.
Journal of molecular biology, 2003
Cooperative binding of ligands to proteins can serve to increase their efficiency and to regulate their activity. Thus, understanding of the mechanism of cooperativity is one of the central concerns of molecular biology. For the tetrameric human hemoglobin (HbA), the cooperative mechanism involves a reasonably well understood combination of tertiary and quaternary changes that occur during the binding process. The dimeric hemoglobin of Scapharca (HbI), which is composed of subunits with the same fold as in HbA, is also highly cooperative but the structural changes on ligand binding are small. A re-orientation of Phe97 in the binding pocket and changes in the number of interfacial water moleculess have been implicated in the cooperative mechanism. To explore the role of these factors, we have investigated models of partially liganded intermediate states of HbI with molecular dynamics simulation methods. Since, unlike HbA, no structures for intermediates are available, they were constructed by combining subunits from the unliganded and liganded dimers. Two structurally distinct intermediates were examined, and it was shown that the transition between the two intermediates is directly coupled to the number of interfacial water molecules. Further, it was found that there is a well-defined water channel that connects the interface between the subunits to bulk water. The bottleneck (gate) of the channel, which can be open or closed, is made of hydrophilic residues. The implication of the present results for the cooperative mechanism of HbI is discussed.
Biophysical Journal, 1998
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-723 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 Ͼ100 cm Ϫ1 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-723 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.
Biophysical Journal, 2005
The results of extended (80-ns) molecular dynamics simulations of wild-type and YQR triple mutant of sperm whale deoxy myoglobin in water are reported and compared with the results of the simulation of the intermediate(s) obtained by photodissociation of CO in the wild-type protein. The opening/closure of pathways between preexistent cavities is different in the three systems. For the photodissociated state, we previously reported a clear-cut correlation between the opening probability and the presence of the photolyzed CO in the proximity of the passage; here we show that in wild-type deoxy myoglobin, opening is almost random. In wild-type deoxy myoglobin, the passage between the distal pocket and the solvent is strictly correlated to the presence/absence of a water molecule that simultaneously interacts with the distal histidine side chain and the heme iron; conversely, in the photodissociated myoglobin, the connection with the bulk solvent is always open when CO is in the vicinity of the A pyrrole ring. In YQR deoxy myoglobin, the mutated Gln(E7)64 is stably H-bonded with the mutated Tyr(B10)29. The essential dynamics analysis unveils a different behavior for the three systems. The motion amplitude is progressively restricted in going from wild-type to YQR deoxy myoglobin and to wild-type myoglobin photoproduct. In all cases, the principal motions involve mainly the same regions, but their directions are different. Analysis of the dynamics of the preexisting cavities indicates large fluctuations and frequent connections with the solvent, in agreement with the earlier hypothesis that some of the ligand may escape from the protein through these pathways.
Journal of Biological Chemistry, 1997
Residue Phe 97 , which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu 97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe 97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe 97 side chain is also associated with diminished cooperativity, since Leu 97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin. The homodimeric hemoglobin (HbI) 1 from the blood clam Scapharca inaequivalvis offers a simple model system for studying communication between two chemically identical subunits. Analysis of the mechanistic details of this intersubunit communication is being pursued to provide insights into the regulation of protein function. Scapharca HbI binds oxygen cooperatively, with a p50 of 7.8 torr and a Hill coefficient (n) of 1.5 at 20°C, neither of which changes as pH varies from 5.5 to 9.0 (1). Although the individual subunits of HbI have the same myoglobin fold as mamma
Journal of Biological Chemistry, 1999
The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6 -9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm ؊1 and shifts to 553 cm ؊1 in H 2 18 O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.