The use of methyl-substituted benzhydroxamic acids as structural probes of peroxidase substrate binding.

Abstract

The interactions between cyanide-ligated horseradish peroxidase (isoenzyme C) and a series of monosubstituted methylbenzhydroxamic acids have been studied using proton NMR techniques, and compared with analogous data for benzhydroxamic acid itself. Use of the methyl-substituted compounds in two-dimensional NOE experiments provides additional data on the structure of the aromatic donor molecule binding site through identification of new NOE connectivities with amino acid side-chains in the heme binding pocket. In this context the methyl group of the substituted benzhydroxamic acids acts as an effective probe of their local environment in the aromatic donor molecule binding site. While all the benzhydroxamic acids interact strongly with the heme edge at heme methyl C18H3, the methyl groups of 3-methylbenzhydroxamic and 4-methylbenzhydroxamic acids principally interact with the distal His42 C epsilon 1H and Phe A side-chains respectively. This confirms that the binding site is situated towards the distal side of the heme plane and that the Phe A side-chain known to participate in aromatic donor binding is located towards the entrance of the site. Dynamic properties of these peroxidase complexes are addressed in terms of ligand exchange phenomena and a comparison made with data available for benzhydroxamic acid. The existence of alternative binding modes in the case of the methylbenzhydroxamic acids is also discussed.