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

Abstract

Using time-resolved X-ray crystallography, we contrast a bifunctional dehaloperoxidase-hemoglobin (DHP) with previously studied examples of myoglobin and hemoglobin in order to understand the functional role of the distal pocket of globins. One key functional difference between the DHP and other globins is the requirement that H2O2 enter the distal pocket of oxyferrous DHP in order to displace O2 from the heme Fe atom and thereby activate the heme for the peroxidase function. The open architecture of DHP permits more than one molecule to simultaneously enter the distal pocket of the protein above the heme in order to facilitate the unique peroxidase cycle starting from the oxyferrous state. The time-resolved X-ray data show that the distal pocket of DHP lacks a protein valve found in the two other globins that have been studied previously. The photolyzed CO ligand trajectory in DHP does not have a docking site. Rather the CO moves immediately to the Xe-binding site. From there CO can escape, but also recombine an order of magnitude more rapidly than in other globins. The contrast with DHP dynamics and function more precisely defines the functional role of the multiple conformational states of myoglobin. Taken together with the high reduction potential of DHP, the open distal site helps to explain how a globin can also function as a peroxidase.

Correlation of Heme Binding Affinity and Enzyme Kinetics of Dehaloperoxidase

“…Increasing the hydrophobicity of the heme pocket 44 increases the binding affinity of the heme and stabilizes a five-coordinate monohistidine adduct, but it is not desirable for peroxidase function. Because the peroxidase function requires protein fluctuations to permit two molecules (H 2 O 2 and O 2 ) simultaneously in the distal pocket, 78 the very increase in hydrophobicity that increases heme binding affinity also would be expected to decrease the access of those molecules to the distal pocket as required for peroxidase function. 8,79 The distal histidine in DHP (H55) is required for both oxygen transport and peroxidase function in DHP.…”

A Model for the Flexibility of the Distal Histidine in Dehaloperoxidase-Hemoglobin A Based on X-ray Crystal Structures of the Carbon Monoxide Adduct

“…Recent studies have shown that DHP can be activated by H 2 O 2 for peroxidase function starting from the oxyferrous state, which makes DHP a unique peroxidase-hemoglobin dual functional enzyme. , The observation of an internal substrate binding site by X-ray crystallography , has also helped to resolve the paradox since it suggests that substrate binding may function as a trigger for the switch from globin to peroxidase function by its strong interaction with the heme Fe. One can identify both an electrostatic component , in addition to a steric effect due to substrate binding. , The observation of a time-resolved X-ray crystal structure is consistent with the open distal pocket in DHP . The distal histidine, H55, is clearly involved in the entrance and exit of the substrate .…”

“… 13 , 14 The observation of a time-resolved X-ray crystal structure is consistent with the open distal pocket in DHP. 15 The distal histidine, H55, is clearly involved in the entrance and exit of the substrate. 16 We have hypothesized that the unusual flexibility of H55 is linked to its role in the triggering the functional switch in DHP, 17 , 18 by analogy to the flavohemoglobins.…”