Nitric oxide and organic ligand complexes of the myoglobin cavity mutant H93G: A model for nitric oxide - heme interaction

When nitric oxide (NO) binds to heme proteins, it exerts a repulsive trans-effect that weakens or breaks the proximal ligand-iron bond. Factors governing the trans-effect are central to understanding the physiological function of NO and were studied using the myoglobin (Mb) cavity mutant, H93G. The H93G mutation replaces the proximal histidine of Mb with glycine, generating a cavity adjacent to the proximal site of the heme iron. This cavity can be filled with small organic ligands (L) to form a 6-coordinate complex, H93G(L)NO, when exposed to NO. Removal of the organic ligand generates a 5-coordinate complex, H93G-NO, in which NO could bind on either the distal or proximal side of the heme. Line broadening of site-specific 19F NMR probes caused by the paramagnetic Fe-NO center indicates that NO binds on the distal side in H93G-NO. This result was confirmed by FTIR measurements of the N-O stretching frequency nu N-O for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of 5- and 6-coordinate MbNO complexes indicate that nuN-O shifts to higher energy for distal, but not proximal mutations relative to wild-type and H93G MbNO. This result suggests that NO binds on the distal side in 5- and 6-coordinate MbNO complexes of H93G.;To examine factors affecting NO binding to H93G, vibrational and kinetic measurements were compared for H93G(L)NO substituted with various organic ligands. Values of nuN-O and nuFe-NO for H93G(L)NO, as determined by FTIR and resonance Raman spectroscopy vary with proximal ligand and are anti-correlated, indicating the presence of pi-backbonding in NO binding to H93G. Comparison of nuN-O and nuFe-NO with ligand binding affinities (KLigand) illustrates the inverse trans-effect for NO binding in H93G(L)NO. Although rates of NO dissociation from H93G(L)NO also vary with organic ligand, temperature dependent parameters do not correlate well with nu Fe-NO, suggesting that NO dissociation involves processes in addition to Fe-NO bond breaking. Finally, results of isothermal titration calorimetry are reported for organic ligand binding to the 5-coordinate met H2O form of H93G. Organic ligand binding to H93G is exothermic, with enthalpy values ranging from -2.8 to -15 kcal/mol, and association constants varying from 2.4 x 104 to 5.0 x 103.