PROBING THE AROMATIC-DONOR-BINDING SITE OF HORSERADISH-PEROXIDASE USING SITE-DIRECTED MUTAGENESIS AND THE SUICIDE SUBSTRATE PHENYLHYDRAZINE

The haem groups from two classes of site-directed mutants of horseradi sh peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]H RP-C and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F 142A]HRP-C and [F143A]HRP-C*) were extracted and analysed by reverse- phase HPLC after phenylhydrazine-induced suicide inactivation. The rel ative abundance of the two covalently modified haems, C20-phenyl (delt a-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topolo gical probe for changes induced in the protein architecture in the vic inity of the haem active site and substrate-access channel. Although d iffering considerably in their efficiency as peroxidases (([H42L]HRP-C exhibited only approximately 0.03 % of the peroxidase activity of wi ld type), the variants studied gave rise to a modification pattern typ ical of an exposed haem edge thereby strengthening the argument that i t is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aroma tic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylati on at the haem C20 position remained remarkably constant. Our findings suggest that the overall efficacy of haem modification catalysed by H RP-C during turnover with phenylhydrazine and its vulnerability toward s inactivation are related to its general ability to bind aromatic don or molecules. Results from phenylhydrazine treatment of HRP-C wild-typ e and mutant variants were compared with those obtained for Coprinus c inereus peroxidase, an enzyme which from its structure is known to hav e a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albei t with a relatively high K-d (K-d 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why phenylhydrazine-treated C. cinereus pe roxidase was more resistant to haem modification and phenyl-radical-ba sed inactivation than HRP-C.