Photolysis of the cyanide adduct of ferrous horseradish peroxidase

AbstractThe mutual interplay between redox- and anion-linked protonation processes in HRP has been investigated. Above pH 7.5, the midpoint potential is pH-independent and the lack of pH-dependency of the dissociation constant, Kd, of the ferrous HRP-cyanide compound shows that both the cyanide anion and the proton are stably bound in the product. Below pH 7.5, Kd increases with decreasing pH and the midpoint potential of the unligated form becomes pH-dependent. These data show that the redox- and anion-linked protonations are mutually exclusive, and are most consistent with protonation of the same residue in the distal heme pocket, His-42, in both cases. Photolysis of the ferroheme-cyanide compound has been investigated and conditions have been identified in which cyanide photolysis is accompanied by the co-migration of a proton, presumably from the protonated His-42. At room temperature, cyanide recombination from solution is a simple second order process with no observable geminate processes occurring on time scales slower than microseconds. However, a dramatic decrease in photolysis yield as temperature is lowered suggests that submicrosecond geminate recombination processes can become dominant. The ferroperoxidase-cyanide system provides a model system for study not only of movement of a more hydrophilic ligand through the protein structure, but also of its associated co-migrating proton