THE PROXIMAL LIGAND VARIANT HIS93TYR OF HORSE HEART MYOGLOBIN

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of repl acing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between p H 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the ac tive site. The EPR spectrum of the variant is that of a high-spin, rho mbic system similar to that reported for bovine liver catalase. The 1D H-1-NMR spectrum of the variant confirms the five-coordinate nature o f the heme iron center and exhibits a broad resonance at 112.5 ppm tha t is attributable to the meta protons of the phenolate ligand. This re sult indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is uncha nged from that of the wild-type protein (pH 8) while the midpoint redu ction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 m V lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal T yr ligand is accommodated by a shift of the F helix (residues 88-99) i n which this residue resides away from the heme pocket. Additional eff ects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hy drogen bonding interactions with the heme prosthetic group. The positi on of the Tyr93 residue with respect to the heme group is also differe nt from that of the His93 residue normally present and resembles that of the proximal Tyr residue of bovine liver catalase.