RECOMBINANT HORSERADISH-PEROXIDASE ISOENZYME-C - THE EFFECT OF DISTALHEME CAVITY MUTATIONS (HIS42-]LEU AND ARG38-]LEU) ON COMPOUND-I FORMATION AND SUBSTRATE-BINDING

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues. h istidine 42 and arginine 38, in the formation of compound I and in sub strate binding. The role of these residues as general acid-base cataly sts originally proposed for cytochrome c peroxidase by Poulos and Krau t in 1980 was assessed for HRPC. Replacement of histidine 42 by leucin e [(H42L)HRPC] decreased the apparent bimolecular rate constant for t he reaction with hydrogen peroxide by five orders of magnitude (k(1) = 1.4 x 10(2) M(-1)s(-1)) compared with both native-glycosylated and re combinant forms of HRPC (k(1) = 1.7 x 10(7) M(-1)s(-1)). The first-ord er rate constant for the heterolytic cleavage of the oxygen-oxygen bon d to form compound I was estimated to be four orders of magnitude slow er for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC ] decreased the observed pseudo-first-order rate constant for the rea ction with hydrogen peroxide by three orders of magnitude (k(1) = 1.1 x 10(4) M(-1)s(-1)), while the observed rate constant of oxygen bond s cission was decreased sixfold (k(2) = 142 s(-1)). These rate constants are consistent with arginine 38 having two roles in catalysing compou nd I formation: firstly, promotion of proton transfer to the group of histidine 42 to facilitate peroxide anion binding to the haem, and sec ondly, stabilisation of the transition slate for the heterolytic cleav age of the oxygen-oxygen build. These roles for arginine 38 explain, i n part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydro xamic acid to (H42L)HRPC and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affini ty.