Dynamics of Nitric Oxide Rebinding and Escape in Horseradish

Studies of the kinetics of diatomic ligand recombination to heme proteins following photodissociation can be used effectively to probe protein dynamics1 and help to reveal the diffusion pathway of the photodissociated ligand (NO, CO, and O2) within the dynamically fluctuating protein matrix. For example, cavities within the protein matrix of Mb, known as xenon pockets (i.e., Xe1–4), have been shown to play a crucial role in the diatomic ligand migration process.2 A variety of other kinetic studies involving ligand rebinding to myoglobin (Mb) and its mutants have been performed to help map out the ligand entrance and exit pathways of this prototypical heme system.1–4 In contrast to the diatomic ligand binding role of Mb, horseradish peroxidase (HRP) is a heme protein with peroxidase activity.5 Although it utilizes the same histidine-ligated heme prosthetic group as Mb to form the functional active site, HRP has a significantly altered distal pocket architecture and plays a very different physiological role than Mb. As a result, it would not be surprising if ligand diffusion inside the protein matrix was quite different. The rebinding of CO to HRP has been studied extensively,6,7 but only a single picosecond kinetics study has been reported,8 which found a relatively small CO geminate amplitud