Rebinding dynamics of NO to microperoxidase-8 probed by time-resolved vibrational spectroscopy

Abstract

Femtosecond vibrational spectroscopy was used to probe the rebinding kinetics of NO to microperoxidase-8 (Mp), an ideal model system for the active site of ligand-binding heme proteins, including myoglobin and hemoglobin, after the photodeligation of MpNO in glycerol/water (G/W) solutions at 294 K. The geminate rebinding (GR) of NO to Mp in viscous solutions was highly efficient and ultrafast and negligibly dependent on the solution viscosity, which was adjusted by changing the glycerol content from 65% to 90% by volume in G/W mixtures. The kinetics of the GR of NO to Mp in viscous solutions was well represented by an exponential function with a time constant of ca. 11 ps. Although the kinetic traces of the GR of NO to Mp in solutions with three different viscosities (18, 81, and 252 cP) almost overlap, they show a slight difference early in the decay process. The kinetic traces were also described by the diffusion-controlled reaction theory with a Coulomb potential. Since the ligand is deligated in a neutral form, an ionic pair of NO⁻ and Mp⁺ may be produced before forming the Mp–NO bond by an electron transfer from Mp to NO as the deligated NO is sufficiently near to the Fe atom of Mp. The strong reactivity between NO and ferrous heme may arise from the Coulomb interaction between the reacting pair, which is consistent with the harpooning mechanism for NO binding to heme.