THE STRETCHING FREQUENCIES OF BOUND ALKYL ISOCYANIDES INDICATE TWO DISTINCT
LIGAND ORIENTATIONS WITHIN THE DISTAL POCKET OF MYOGLOBIN
George C. Blouin, Edwin J. Heilweil, Angela R. Hight
Alkyl isocyanides (CNRs) have a long history of use as probes of steric constraints in the binding pockets of myoglobins (Mb) and hemoglobins (Hb). However, little is known about their conformations within those environments. The FTIR spectra of CNRs bound to sperm whale Mb have VCN bands at ~2075 cm-1 and ~2125 cm-1 that have been assigned to in and out conformations, respectively. In the in conformation, the ligand points toward the protein interior, and the lower VCN results from donation of a hydrogen bond from the distal His64(E7) to the bound isocyano group. In the out conformation, the ligand displaces the His64 side chain into solvent and away from the binding site. Support for this interpretation includes: (1) the similar dependence of VCO on the His64 conformation in MbCO; (2) the absence of the low-frequency VCN peak for CNRs bound in the apolar binding sites of H64A Mb, H64L Mb, and micelles containing model heme; and (3) a correlation between the fraction of photodissociated CNRs that are held in the binding pocket and rebind geminately over 100s of ns and the fraction of CNRs in the in conformation. CNRs that point out through an open His64 “gate” rapidly escape following dissociation from the heme iron.
The situation is more complex for CNRs bound to isolated subunits of human Hb. The FTIR spectra contain three VCN bands, and geminate rebinding followed at visible wavelengths occurs in multiple phases. To interpret these data, we are currently studying CNRs bound to Mb, Hb, and a heme/micelle model system by Raman spectroscopy to determine the dependence of VCN on Fe-CNR back bonding, and by time-resolved IR to assign the rebinding phases to their respective VCN bands.