Mechanistic Studies on the Reversible Binding of Nitric Oxide to Metmyoglobin

The ferriheme protein metmyoglobin (metMb) in buffer solution at physiological pH 7.4 reversibly binds the biomessenger molecule nitric oxide to yield the nitrosyl adduct (metMb(NO)). The kinetics of the association and dissociation processes were investigated by both laser flash photolysis and stopped-flow kinetics techniques at ambient and high pressure, in three laboratories using several different sources of metMb. The activation parameters ΔH ⧧, ΔS ⧧, and ΔV ⧧ were calculated from the kinetic effects of varying temperature and hydrostatic pressure. For the “on” reaction of metMb plus NO, reasonable agreement was found between the various techniques with ΔH on ⧧, ΔS on ⧧, and ΔV on ⧧ determined to have the respective values ∼65 kJ mol-1, ∼60 J mol-1 K-1, and ∼20 cm3 mol-1. The large and positive ΔS ⧧ and ΔV ⧧ values are consistent with the operation of a limiting dissociative ligand substitution mechanism whereby dissociation of the H2O occupying the sixth distal coordination site of metMb must precede formation of the Fe−NO bond. While the activation enthalpies of the “off” reaction displayed reasonable agreement between the various techniques (ranging from 68 to 83 kJ mol-1), poorer agreement was found for the ΔS off ⧧ values. For this reason, the kinetics for the “off” reaction were determined more directly via NO trapping experiments, which gave the respective activation parameters ΔH off ⧧ = 76 kJ mol-1, ΔS off ⧧ = ∼41 J mol-1 K-1, and ΔV off ⧧ = 20 cm3 mol-1), again consistent with a limiting dissociative mechanism. These results are discussed in reference to other investigations of the reactions of NO with both model systems and metalloproteins.