Protein conformation-controlled rebinding barrier of NO and its binding trajectories in myoglobin and hemoglobin at room temperature.

The effect of the solvent viscosity on the dynamics of NO rebinding to myoglobin (Mb) and hemoglobin (Hb) was examined by femtosecond (fs) time-resolved vibrational spectroscopy after photodeligation of NO from MbNO and HbNO in various viscous solutions at 283 K using a 580 nm excitation pulse. The rebinding kinetics of NO to both Mb and Hb were nonexponential, but their dependence on the solvent viscosity was different. The rate of NO rebinding to Mb increased with increasing solution viscosity, which was achieved by increasing the glycerol content in glycerol/water mixture. In contrast, the rate of NO rebinding to Hb was independent of the solution viscosity but faster than the fastest rate of NO rebinding observed in Mb. The dynamics of conformational relaxation of the protein after deligation were also measured by probing the evolution of the amide band. The effect of the solvent viscosity on the kinetics of conformational relaxation in both proteins was also quite different. The conformational relaxation of Mb became slower with increasing solution viscosity. On the other hand, the conformational relaxation of Hb was independent of the solution viscosity but slower than the slowest kinetics of Mb. The inverse correlation in the kinetics of conformational relaxation and NO rebinding suggests that the barrier of NO rebinding increases as the conformation of the protein relaxes toward the deligated structure after NO dissociation. The rebinding kinetics of NO to both proteins was well described by a kinetic model incorporating a time-dependent barrier for rebinding and exponential translocations between three states for dissociated NO.