A THEORETICAL-STUDY OF BENZHYDROXAMIC ACID-BINDING MODES IN HORSERADISH-PEROXIDASE

In this study, the substrate binding sites and mode of binding of benz hydroxamic acid (BHA) in the low-spin cyanide-ligated form of horserad ish peroxidase isoenzyme C (HRP-C) have been identified and characteri zed using the X-ray crystallographic structure of HRP-C in the substra te-free form, in combination with the programs AUTODOCK and AMBER. Two criteria were used to select the most favorable binding site: the int eraction energy of BHA with the protein and the mobility of BHA in eac h binding site. Using these criteria, the binding site located on the distal side of the heme and surrounded by His42, Arg38, Pro139, Leu138 , Ala140, Phe68, Pro141, Gly69, Phe179, Phe41, Asn70, and Ser73 was fo und most promising. Computed distances between atoms in BHA and atoms in residues of HRP-C/CN were, in general, in good agreement with a sub set of corresponding distances derived from H-1 NMR data. In addition, two strong H bonds of BHA, with Arg38 and the N atom of the cyanide l igand, and two polar interactions of BHA, with His42 and Pro139, were found, consistent with the relatively high binding affinity of BHA for HRP-C/CN. The second most favorable binding site identified was locat ed toward the proximal side of the heme at a distance of only 10 Angst rom from the first binding site, as described above, suggesting it as a temporary storage place for the radical produced by oxidation of the first substrate molecule. This small displacement would allow accommo dation of a second substrate molecule in the first site and dimerizati on to occur after the second substrate radical is formed through a sec ond one-electron oxidation step. Although it is not known whether BHA forms dimers due to oxidation by HRP-C, other phenolic substrates that do form dimers may occupy both the primary binding site, where oxidat ion occurs, and the radical holding site, to facilitate dimer formatio n.