STRUCTURAL INTERACTIONS BETWEEN HORSERADISH-PEROXIDASE-C AND THE SUBSTRATE BENZHYDROXAMIC ACID DETERMINED BY X-RAY CRYSTALLOGRAPHY

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromati c binding pocket. The crystal structure of the peroxidase-substrate co mplex has been determined to 2.0 Angstrom resolution with a crystallog raphic R-factor of 0.176 (R-free = 0.192). A well-defined electron den sity for BHA is observed in the peroxidase active site, with a hydroph obic pocket surrounding the aromatic ring of the substrate. The hydrop hobic pocket is provided by residues H42, F68, G69, A140, P141, and F1 79 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 Angstrom) found between heme C18-methyl and BHA C63, Very little struc tural rearrangement is seen in the heme crevice in response to substra te binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 Angstrom toward the heme iron. The bo und BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 Angstrom above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible, Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrop hobic pocket, suggests that BHA is a general counterpart for the conse rved hydrogen bond donors and accepters of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase sub strates.