An essential role of active site arginine residue in iodide binding and histidine residue in electron transfer for iodide oxidation by horseradish peroxidase

The objective of the present study is to delineate the role of active site arginine and histidine residues of horseradish peroxidase (HRP) in controll ing iodide oxidation using chemical modification technique. The arginine sp ecific reagent, phenylglyoxal (PGO) irreversibly blocks iodide oxidation fo llowing pseudofirst order kinetics with second order rate constant of 25.12 min(-1) M-1. Radiolabelled PGO incorporation studies indicate an essential role of a single arginine residue in enzyme inactivation. The enzyme can b e protected both by iodide and an aromatic donor such as guaiacol. Moreover , guaiacol-protected enzyme can oxidise iodide and iodide-protected enzyme can oxidise guaiacol suggesting the regulatory role of the same active site arginine residue in both iodide and guaiacol binding. The protection const ant (K-p) for iodide and guaiacol are 500 and 10 muM respectively indicatin g higher affinity of guaiacol than iodide at this site. Donor binding studi es indicate that guaiacol competitively inhibits iodide binding suggesting their interaction at the same binding site. Arginine-modified enzyme shows significant loss of iodide binding as shown by increased K-d value to 571 m M from the native enzyme (K-d = 150 mM). Although arginine-modified enzyme reacts with H2O2 to form compound II presumably at a slow rate, the latter is not reduced by iodide presumably due to low affinity binding. The role of the active site histidine residue in iodide oxidation was also studied after disubstitution reaction of the histidine imidazole nitrogens with diethylpyrocarbonate (DEPC), a histidine specific reagent. DEPC blocks iodide oxidation following pseudofirst order kinetics with second order ra te constant of 0.66 min(-1) M-1. Both the nitrogens (delta, epsilon) of his tidine imidazole were modified as evidenced by the characteristic peak at 2 22 nm. The enzyme is not protected by iodide suggesting that imidazolium io n is not involved in iodide binding. Moreover, DEPC-modified enzyme binds i odide similar to the native enzyme. However, the modified enzyme does not f orm compound II but forms compound I only with higher concentration of H2O2 suggesting the catalytic role of this histidine in the formation and autor eduction of compound I. Interestingly, compound I thus formed is not reduce d by iodide indicating block of electron transport from the donor to the co mpound I. We suggest that an active site arginine residue regulates iodide binding while the histidine residue controls the electron transfer to the h eme ferryl group during oxidation.