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.
Falconi
M
M
Department of Biology and INFM, University of Rome "Tor Vergata," 00133 Roma, Italy.
Desideri
A
A
Cupane
A
A
Leone
M
M
Ciccotti
G
G
Peterson
E S
ES
Friedman
J M
JM
Gambacurta
A
A
Ascoli
F
F
D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism; D019281:Dimerization; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D008974:Mollusca / Q000737:chemistry*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013696:Temperature; D014867:Water / Q000737:chemistry
D006454:Hemoglobins; D014867:Water; C083082:dimeric hemoglobin, Scapharca inaequivalvis; D006418:Heme; D002248:Carbon Monoxide; C100003:deoxyhemoglobin