Distal Histidine Flexibility as the Key to the Reactivity of Dehaloperoxidase-Hemoglobin

Abstract

The enzymes dehaloperoxidase A and B (DHP A&B) from the marine worm Amphitrite ornata are unique hemoglobins that function as peroxidases, capable of converting 2,4,6-trihalo- phenols into the corresponding 2,6-dihalogenated quinones. It is difficult to explain the peroxidase activity of DHPs A&B since they are clearly both hemolgobins. Our recent discovery of an internal inhibitor binding site, in the distal pocket, clearly establishes the peroxidase function and suggests that the distal histidine (H55) plays the central role in both function switching and activation of DHPs A&B. Similar to many myoglobins, H55 of DHP A is observed in two conformations, open and closed. However, in DHP A, H55 has been shown to exist in the open conformation to a much greater extent than in any known myoglobin. This results in an open exit channel for bound diatomic ligands, which leads to rapid escape. However, the open conformation may also permit the entry of hydrogen peroxide and even the native inhibitor, 4-bromophenol. We also have structural and spectroscopic evidence that external binding of substrate pushes H55 more deeply into the protein. This may serve to aid in the activation of bound hydrogen peroxide as part of the peroxidase mechanism. Thus, in DHP A, the closed and open states are inactive and active, respectively, for peroxidase chemistry. We have systematically probed this hypothesis using a variety of methods and site-directed mutants to determine the role played by the distal cavity in the stabilization of the inhibitor and the flexibility of the distal histidine. Based on the binding of the series of 4-halophenols in X-ray crystal structures, it is evident that there is a protein cavity deep inside DHP A. The functional relevance of this cavity will be elucidated.