Photolysis of the cyanide adduct of ferrous horseradish peroxidase

The 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, K-d, 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, K-d 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.