Structural evidence for stabilization of inhibitor binding by a protein cavity in the dehaloperoxidase‐hemoglobin from Amphitrite ornata

Abstract

A functional role for a protein cavity that stabilizes inhibitor binding has been established based on a comparison of Xe-derivatized and inhibitor-bound X-ray crystal structures in dehaloperoxidase-hemoglobin (DHP A) of Amphitrite ornata. The internal binding affinity of four different inhibitors, 4-fluorophenol, 4-chlorophenol, 4-bromophenol, and 4-iodophenol in the distal pocket has been shown previously to increase proportional to the radius of the para-halogen atom. Inhibition of oxidation of the native substrate, 2,4,6-tribromophenol, has been shown to follow the trend in inhibitor binding strength, because of a two-site competitive inhibition mechanism that involves displacement of the substrate by the inhibitor in a gated mechanism involving the distal histidine of DHP A. In this study, it is shown that the origin of the stronger binding by a larger para-halogen substituent coincides structurally with a Xe-binding cavity (Xe1) characterized structurally by X-ray crystallography. The Xe1 site is surrounded by amino acid resides L100, F21, F24, F35, F60, and V59 in the distal pocket, located 4.8 Å from the heme iron, in a position that is coincident with the para-bromine atom of the inhibitor 4-bromophenol. 4-bromophenol is prevalent in benthic ecosystems where A. ornata resides. A second, less well-defined, binding site in DHP A, labeled as Xe2, is located near the surface of the protein in the vicinity of amino acid residues L62, R69, D79, T82, and L83, which may be related to substrate docking on the surface of DHP A.

Distinct Enzyme–Substrate Interactions Revealed by Two Dimensional Kinetic Comparison between Dehaloperoxidase-Hemoglobin and Horseradish Peroxidase

“…The substrate inhibition produced by 2,4,6-TXP (X = Cl, Br) is presumably due to the internal binding in the distal pocket in a position that is observed in the crystal structure, 15,17 in which the substrate either blocks the H 2 O 2 heme iron axial position for H 2 O 2 to form compound 0 or impedes the entering of H 2 O 2 into the distal pocket.…”

“…[15][16][17][18][19][20][21]29,30 The evidence for multiple binding sites can be divided into internal sites (X-ray crystallography, NMR; 15−21,30 and flow-EPR, kinetics). 29 The first X-ray crystal structure of DHP showed that 4-iodophenol (4-IP) binds internally in the distal pocket above the heme.…”

Functional Consequences of the Open Distal Pocket of Dehaloperoxidase-Hemoglobin Observed by Time-Resolved X-ray Crystallography

“…Unlike SWMb, which has 4 Xe binding sites, there is only one identifiable Xe binding site inside DHP (Xe1). (13) Although the result in Figure 1 may appear similar to the trajectories in SWMb*CO (2–3, 34), there are major differences, which are related to the difference in function of DHP. The open distal pocket of DHP, which permits the binding of molecules as large as 2,4,6-tribromophenol (2,4,6-TBP) inside the globin (35) means that there is less hindrance to ligand escape, but also less hindrance for ligand entry and binding to the heme Fe.…”

“…There are four Xe binding sites observed in SWMb, with Xe1 site close to fully occupied in the X-ray structure, and sites Xe2-Xe4 occupied by Xe at 40–50% (36). In DHP only one internal Xe binding site is present, occupied at ~40%, with a second site that has only ~10% occupation located on the surface of the protein (13). The DHP Xe1 binding site is above the heme and coincides with the CO density at 100 ps (Figure 3A).…”

“…During the simulation, the free CO molecule was observed to dwell in the Xe1 binding site for the majority of the simulation time. The distances of the Xe1 binding site in the DHP structure (PDB 3MOU) (13) to the α-carbon of each of the surrounding amino acids are shown as the dotted black lines in the figures. The δ-His tautomer shows more variation in the CO position, which is divided between the Xe binding site and a site in the distal pocket near the heme Fe.…”