PHOTOLYSIS OF THE CYANIDE ADDUCT OF FERROUS HORSERADISH-PEROXIDASE

The mutual interplay between redox- and anion-linked protonation proce sses in HRP has been investigated. Above pH 7.5, the midpoint potentia l 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 p H 7.5, K-d increases with decreasing pH and the midpoint potential of the unligated form becomes pH-dependent. These data show that the redo x- and anion-linked protonations are mutually exclusive, and are most consistent with protonation of the same residue in the distal heme poc ket, His-42, in both cases. Photolysis of the ferroheme-cyanide compou nd has been investigated and conditions have been identified in which cyanide photolysis is accompanied by the co-migration of a proton, pre sumably from the protonated His-42. At room temperature, cyanide recom bination from solution is a simple second order process with no observ able geminate processes occurring on time scales slower than microseco nds. However, a dramatic decrease in photolysis yield as temperature i s lowered suggests that submicrosecond geminate recombination processe s can become dominant. The ferroperoxidase-cyanide system provides a m odel system for study not only of movement of a more hydrophilic ligan d through the protein structure, but also of its associated co-migrati ng proton.