Myoglobin-NO at low pH: free four-coordinated heme in the protein pocket.

In either sperm whale or horse heart myoglobin, binding of NO and lowering of solution pH work together to weaken, and ultimately break, the bond between iron and the proximal histidine. This is reminiscent of the reaction observed at neutral pH in the case of guanylate cyclase, the heme enzyme that catalyzes the conversion of GTP to cGMP. Bond breaking is characterized by a spectral change from a nine-line to a three-line ESR signal and accompanied by a shift from 420 to 387 nm in the UV-vis spectrum of the Soret band maximum. Analysis of the pH-dependent spectral changes shows that they are reversible, at least within a few hours, that the transition is cooperative, involving six protons during pH lowering but only two as it is raised, and that the pK is about 4.7. Different proteins exhibit different pK values, which are generally lower than that for "chelated" protoheme. The pK differences reflect the extra bond stability afforded by the protein structure. Investigations of thermal and photochemical NO displacement by CO suggest that the local pocket around the ligand, although significantly altered (according to circular dichroism investigations), nonetheless still imposes a barrier against the outward diffusion of ligand into the solvent. Nanosecond and picosecond flash photolysis shows that in proteins at low pH there is an extremely efficient geminate recombination of the ligand with the four-coordinated species through a single-exponential process. This occurs to a significantly larger extent than for the case of NO-"chelated" protoheme (where no distal barrier for ligand is present). At neutral pH, when the proximal histidine bond is intact, the geminate recombination for NO takes longer and displays multiexponential kinetics. Altogether, these results suggest that, even though distal effects probably also play a role, proximal effects make an important contribution in modulating ligand-iron bond formation.