FTIR and Resonance Raman Studies of Nitric Oxide Binding to H93G Cavity Mutants of Myoglobin

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe−NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N−O vibrational frequency ν̄N- O for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that ν̄N - O shifts (by as much as 26 cm-1) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while ν̄N - O is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe−NO vibrational frequency ν̄Fe - N O as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that ν̄Fe - N O is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that ν̄N - O and ν̄Fe - N O are not quantitatively correlated for the MbNO complexes presented here. Furthermore, ν̄N - O and ν̄Fe - N O do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of π-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket.