Experimental and Computational Study of the Monomer-Dimer Equilibrium in Dehaloperoxidase from Amphitrite Ornata

Abstract

The enzyme dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a unique hemoglobin that functions as a peroxidase, capable of converting 2,4,6-trihalophenols (TBP, TCP, and TFP) into dihalogenated quinones and other products. The DHP protein crystallizes as a dimer. Yet, it was originally characterized as a monomer in solution. We have conducted small angle X-ray scattering (SAXS) in order to probe the monomer-dimer equilibrium in solution. The interest in this area arises from the fact that many hemoglobins are multimers that play an essential role in the cooperativity of oxygen uptake and release. For example, A. ornata possesses a giant hemoglobin (erythrocruorin) like many other marine organisms. Since there are only two known hemoglobin genes (DHP A and DHP B) in A. ornata, it is logical that one or both of these proteins associate with other proteins to form the giant hemoglobin. On the other hand, coelomic DHP does not appear to have a high degree of cooperativity. Moreover, the dimer interface in the X-ray crystal structure of both DHP A and DHP B consists of only 3 amino acid residues. The SAXS data show that the equilibrium for DHP favors the monomer form up to the highest concentrations studied (∼ 200 micromolar). However, is there a small amount of the dimer in solution. Thus, it is of interest to apply the known interfaces from study of the X-ray crystal structure to determine which surfaces of DHP may be interacting weakly in solution. We studied the monomer-dimer interface using molecular dynamics (MD) simulations in order to ascertain the relative strength of these interfaces. These results are used to develop a systematic approach to characterization of monomer-multimer equilibria based SAXS and X-ray crystallography data.