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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 aromatic binding pocket. The crystal
structure of the peroxidase-substrate complex has been determined to 2.0 A
resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A
well-defined electron density for BHA is observed in the peroxidase active site,
with a hydrophobic pocket surrounding the aromatic ring of the substrate. The
hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179
and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found
between heme C18-methyl and BHA C63. Very little structural rearrangement is
seen in the heme crevice in response to substrate binding. F68 moves to form a
lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward
the heme iron. The bound BHA molecule forms an extensive hydrogen bonding
network with H42, R38, P139, and the distal water molecule 2.6 A 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 hydrophobic pocket, suggests that BHA is
a general counterpart for the conserved hydrogen bond donors and acceptors 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 substrates.
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