The proximal ligand variant His93Tyr of horse heart myoglobin

Abstract

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing 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 pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV 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 Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

“…An additional unexchangeable signal, detectable only upon increasing the temperature to 318 K, was observed at 84.1 ppm. Attribution of these two resonances to meta protons of the tyrosine ligand is consistent with previous literature data on catalase (39) and on the H93Y mutant of myoglobin (40). Increasing the pH results in a small shift of all resonances (Fig.…”

“…Tyr is a relatively weak ligand for iron(III) (40,44), and its presence is not vital for the protein fold. In the Y75A mutant loop L2 remains connected to L1 through the heme thanks to the presence of a water molecule (or of His 83 at high pH) in place of Tyr 75 , as depicted in Scheme 1B.…”

Measurement of the Heme Affinity for Yeast Dap1p, and Its Importance in Cellular Function

“…The reduction potential of Dap1p was measured to be −307 mV vs. SHE which is comparable to the −260 mV value of bovine catalase, whose axial ligand is also a tyrosinate (29,30). This value is in the range of cytochrome P 450 reductases,(34) making it tempting to suggest that Dap1p could provide electrons to Erg11p and hence increase the Erg11p activity in the cell with Dap1p present, as suggested by Hughes et al (8).…”

Interaction between Substrate and Oxygen Ligand Responsible for Effective O–O Bond Cleavage in Bovine Cytochrome P450 Steroid 21-Hydroxylase Proved by Raman Spectroscopy

“…5B. Because the ␦(C ␤ C a C b ) mode is sensitive to the hydrogen bonding interaction between heme propionate groups and surroundings (31,32), the substrate-induced downshift of the ␦(C ␤ C a C b ) mode by 5 cm Ϫ1 indicates that the substrate could alter the environment around the heme peripheral groups in the ferrous state. The substrate-induced frequency shifts were also detected in the ␦(C ␤ C a C b ) mode.…”