Effect of H55D Mutation on Kinetics and Structure of Dehaloperoxidase-Hemoglobin A

Abstract

The H55D mutant of dehaloperoxidase-hemoglobin A (DHP A) has been prepared to mimic the H64D mutant of Sperm Whale myoglobin (SWMb), which has the highest peroxidase activity of any myoglobin mutant studied to date. The high peroxidase activity has been attributed to the rapid formation as well as the prolonged lifetime of compound I following addition of H2O2. However, unlike H64D SWMb, the H55D mutant of DHP A has ∼13-fold lower peroxidase activity towards oxidation of 2,4,6-trichlorophenol (TCP) into 2,6-dichloroquinone (DCQ) in the presence of H2O2. The origin of the lower rate constant may be the solvent-exposed conformation of D55, which has the effect of removing the acid-base catalyst necessary for heterolytic cleavage O-O bond in Fe-bound H2O2. These observations further support the hypothesis that the flexibility of distal histidine in wild-type DHP A, plays a crucial role in enzyme function. Remarkably, the H55D mutant also shows inhibition by 4-bromophenol (4-BP) in the oxidation of TCP, which is substantiated by the X-ray crystal structure of 4-BP bound to the internal inhibitor binding site observed in wild type DHP A and B. The inhibitor complex is an unusual internally bound molecule, completely surrounded by the protein. Compound ES, a tyrosine radical species associated with the oxoferryl intermediate detected in DHP A, is not observed in H55D. Thus, the H55D mutant is a key test case for the role of electron transfer in the DHP A mechanism.