Solution ¹H NMR Investigation of the Heme Cavity and Substrate Binding Site in Cyanide-Inhibited Horseradish Peroxidase

Abstract

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.

Application of the paramagnetic dipole field for solution NMR active site structure determination in low‐spin, cyanide‐inhibited ferric hemoproteins

“…The differences in the direction (a) of the Fe-CN tilt between the magnetic axes obtained earlier (38) using either the homology model CcP, or the appropriate HRP crystal structure (39), are within the experimental uncertainty of +108. The difference in the tilt magnitude, b, of 98 on the other hand, is well outside the experimental uncertainty of +18.…”

“…The dispositions of key active site residues in HRP are shown (36) in Figure 13. Yeast cytochrome c peroxidase, CcP, yielded the first crystal structure (37), while horseradish peroxidase, HRP, because of its much greater stability, yielded the more detailed NMR assignment and solution NMR characterization (1,15,38,39). The cyanide-inhibited complex mimics the catalytically active compound-II of heme peroxidases (40).…”

“…Peaks are labeled in the Fischer notation for the heme, and by the one letter code for amino acid residues. Vertical dashed lines connect a given proton in the two complexes (39).…”

“…Hence the determined orientation of the z-axis or Fe-CN vector is the same relative to the protein matrix using the alternate HRP and CcP crystal structures. The tilt (*128) in the FeCN unit in HRP-CN in the direction of the d-meso position was attributed (39) to the formation of a very strong H-bond (40) to the distal His42 N e H.…”

Sequential affinity purification of peroxidase tagged bispecific anti-SARS-CoV antibodies on phenylboronic acid agarose

“…The challenge of obtaining bispecific immunoprobes with high specific activity and purity was circumvented by using benzhydroxamic acid agarose to purify the antibodies [15] tagged with HRPO as a preformed immune complex [15,16]. Benzhydroxamic acid agarose (BHA-agarose) has been reported to bind at the active site of HRPO [17]. Unfortunately, due to the discontinuation of BHA-agarose production by the vendor, we attempted to develop an alternative affinity chromatography method for the purification of BsMAb with one arm specific to HRPO and the other arm specific to Severe Acute Respiratory Syndrome's (SARS-CoV) nucleoprotein (NP) antigen, using novel boronate affinity matrix called m-aminophenylboronic acid agarose (APBA-agarose).…”