Dynamics of Multifunctional Dehaloperoxidase Hemoglobin

Multi-functional dehaloperoxidase-hemoglobin (DHP) is a versatile protein that functions as an oxygen transporter, but also has functions related to detoxification of a range of substrates including brominated phenols and indoles. It is ironic that the inhibitor binding site has been known for 15 years, but the substrate binding sites remain elusive. Two recent X-ray crystallographic studies reveal internal substrate binding sites. However, since there are at least three binding sites for 2,4,6-trihalophenols alone, the functional location of substrate binding remains unresolved. Moreover, recent kinetic data show that a number of indoles are excellent substrates for DHP and the isotope data indicate that some of these are oxidized directly by bound H2O2 (peroxygenase function) or O2 (monooxygenase function). Clearly the internal binding requires a large binding cavity and protein dynamics that permit the entrance of these large molecules into the distal pocket. Time-resolved X-ray crystallography reveals ballistic motion of carbon monoxide to the only internal Xe-binding site and rapid escape from the protein, both features that are consistent with the open architecture of the distal pocket. Preliminary NMR dynamic studies show that this method can be used to complement X-ray crystallography to provide information on the location of substrate binding. Resonance Raman studies complement these methods as well providing a means to study how ligand binding to the heme Fe interacts with the internally bound inhibitors and substrates. This combination of methods has the potential to reveal functional differences in the binding of different substrates and the role played by protein dynamics in controlling the functional switching of DHP.