MYOGLOBIN NO AT LOW PH - FREE 4-COORDINATED HEME IN THE PROTEIN POCKET

In either sperm whale or horse heart myoglobin, binding of NO and lowe ring 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 bre aking is characterized by a spectral change from a nine-line to a thre e-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-depende nt spectral changes shows that they are reversible, at least within a few hours, that the transition is cooperative, involving six protons d uring pH lowering but only two as it is raised, and that the pK is abo ut 4.7. Different proteins exhibit different pK values, which are gene rally lower than that for ''chelated'' protoheme. The pK differences r eflect the extra bond stability afforded by the protein structure. Inv estigations of thermal and photochemical NO displacement by CO suggest that the local pocket around the ligand, although significantly alter ed (according to circular dichroism investigations), nonetheless still imposes a barrier against the outward diffusion of ligand into the so lvent. Nanosecond and picosecond flash photolysis shows that in protei ns at low pH there is an extremely efficient geminate recombination of the ligand with the four-coordinated species through a single-exponen tial process. This occurs to a significantly larger extent than for th e case of NO-''chelated'' protoheme (where no distal barrier for ligan d is present). At neutral pH, when the proximal histidine bond is inta ct, the geminate recombination for NO takes longer and displays multie xponential kinetics. Altogether, these results suggest that, even thou gh distal effects probably also play a role, proximal effects make an important contribution in modulating ligand-iron bond formation.