STRUCTURAL AND DYNAMIC PROPERTIES OF THE HOMODIMERIC HEMOGLOBIN FROM SCAPHARCA-INAEQUIVALVIS THR-72-]IIE MUTANT - MOLECULAR-DYNAMICS SIMULATION, LOW-TEMPERATURE VISIBLE ABSORPTION-SPECTROSCOPY, AND RESONANCE RAMAN-SPECTROSCOPY STUDIES

Molecular dynamics simulations, low temperature visible absorption spe ctroscopy, and resonance Raman spectroscopy have been performed on a m utant of the Scapharca inaequivalvis homodimeric hemoglobin, where res idue threonine 72, at the subunit interface, has been substituted by i soleucine. Molecular dynamics simulation indicates that in the Thr-72- ->Ile mutant several residues that have been shown to play a role in l igand binding fluctuate around orientations and distances similar to t hose observed in the x-ray structure of the CO derivative of the nativ e hemoglobin, although the overall structure remains in the T state. V isible absorption spectroscopy data indicate that in the deoxy form th e Soret band is less asymmetric in the mutant than in the native prote in, suggesting a more planar heme structure; moreover, these data sugg est a similar heme-solvent interaction in both the liganded and unliga nded states of the mutant protein, at variance with that observed in t he native protein. The ''conformation sensitive'' band III of the deox y mutant protein is shifted to lower energy by >100 cm(-1) with respec t to the native one, about one-half of that observed in the low temper ature 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 de oxy 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 los s of water in the heme pocket. In line with this latter interpretation , the number of water molecules that dynamically enters the intersubun it interface, as calculated by the molecular dynamics simulation, is l ower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal hist idine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent w ith a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen af finity displayed by the Thr-72-->Ile mutant is mainly due to a local p erturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate intera ctions. These changes are consistent with a destabilization of the T s tate and a stabilization of the R state in the mutant relative to the native protein.