A photolysis-triggered heme ligand switch in H93G myoglobin

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR ) spectroscopy have been used to identify the ligation state of ferrous hem e iron for the H93G proximal cavity mutant of myoglobin in the absence of e xogenous ligand on the proximal side. Preparation of the H93G mutant of myo globin has been previously reported for a variety of axial ligands to the h eme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatu r, S. M,, Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981- 6982]. The present study examines the ligation states of heme in preparatio ns of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93 G, resonance Raman spectroscopic evidence shows water to be the axial (fift h) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe -C stretching frequencies for the CO adduct indicates that it is six-coordi nate with a histidine trans ligand. Following photolysis of CO, a time-depe ndent change in ligation is evident in both step-scan FTIR and saturation r esonance Raman spectra, leading to the conclusion that a conformationally d riven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine l igation, while conformational strain favors the dissociation of histidine f ollowing photolysis of CO. The replacement of histidine by water in the fiv e-coordinate complex is estimated to occur in <5 mus. The results demonstra te that the H93G myoglobin cavity mutant has potential utility as a model s ystem for studying the conformational energetics of ligand switching in hem e proteins such as those observed in nitrite reductase, guanylyl cyclase an d possibly cytochrome c oxidase.