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Hemoglobin/dehaloperoxidase (DHP) from Amphitrite ornata, is the first discovered globin which in addition to the oxygen binding has an additional physiological function as an enzyme. In the ferric state it is a dehaloperoxidase, which dehalogenates a wide range of halophenols. DHP evolved from an ancestral oxygen carrier and acquired the peroxidase function in response to environmental pressures. The peroxidase activity of DHP is much higher than that of vertebrate myoglobins (Mb) yet lower than that of typical peroxidases. Also, DHP exhibits about 10-fold lower oxygen affinity than Mb.
The first aim of this research was to identify the halophenolic substrates binding sites by determining the crystal structures of complexes of DHP with a substrate 2,4,6-trichlorophenol (TCP). Two mutually exclusive TCP binding modes were observed in the crystal structures of DHP mutants. They provided important implications for the DHP catalytic mechanism. The substrates binding order is the same as in classical peroxidases: hydrogen peroxide binds first leading to the formation of Compound I and only this intermediate binds halophenolic substrates in the productive manner. The binding of halophenols to DHP, prior to the formation of Compound I, prevents the approach of hydrogen peroxide to the heme and is inhibitory. This model was confirmed by our observation that higher substrate concentrations are inhibitory.
The second aim of this research was to study the functional and structural properties of two closely related DHP isoenzymes: DHP A and DHP B which differ in only five amino acids. The first approach was to investigate functional and structural differences, based on the analysis of transition mutants from DHP A to DHP B (Y34N DHP A and Y34NS/91G DHP A). The second approach is to study the "DHP A-like" K42Y Mb mutant and "DHP B-like" K42N Mb mutant, which mimic the heme environment at position 34 in DHPs. These studies suggest the roles of amino acids at positions 34 and 91 in the variation of functional properties between DHP A and DHP B.
The third part of this thesis describes the binding of phenol, a relatively big ligand, in the proximal cavity. The kinetic studies showed that phenol acts both as a competitive inhibitor likely interfering with the substrate binding at the heme edge and as a weak activator, likely through binding in the proximal cavity.