X-ray crystal structures of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata soaked with substrate, 2,4,6-tribromophenol (2,4,6-TBP), in buffer solvent with added methanol (MeOH), 2-propanol (2-PrOH), and dimethyl sulfoxide (DMSO) reveal an internal substrate binding site deep in the distal pocket above the α-edge of the heme that is distinct from the previously determined internal inhibitor binding site. The peroxidase function of DHP A has most often been studied using 2,4,6-trichlorophenol (2,4,6-TCP) as a substrate analogue because of the low solubility of 2,4,6-TBP in an aqueous buffer solution. Previous studies at low substrate concentrations pointed to the binding of substrate 2,4,6-TCP at an external site near the exterior heme β- or δ-edge as observed in the class of heme peroxidases. Here we report that the turnover frequencies of both substrates 2,4,6-TCP and 2,4,6-TBP deviate from Michaelis-Menten kinetics at high concentrations. The turnover frequency reaches a maximum in the range of 1400-1700 μM, with a decrease in rate at higher concentrations that is both substrate- and solvent-dependent. The X-ray crystal structure is consistent with the presence of an internal active site above the heme α-edge, in which the substrate would be oxidized in two consecutive steps inside the enzyme, followed by attack by H2O via a water channel in the protein. The physiological role of the internal site may involve interactions with any of a number of aromatic toxins found in benthic ecosystems where A. ornata resides.

Chemical and thermal denaturation of dehaloperoxidase-hemoglobin (DHP) was investigated to test the relative stability of isoforms DHP A and DHP B and the H55V mutant of DHP A with respect to heme loss. In thermal denaturation experiments, heme loss was observed at temperatures of 54, 46, and 61 °C in DHP A, DHP B, and H55V, respectively. Guanidinium hydrochloride (GdnHCl)- and urea-induced denaturation was observed at respective concentrations of 1.15 ± 0.01 M DHP A and 1.09 ± 0.02 M DHP B, and 5.19 ± 0.05 M DHP A and 4.12 ± 0.14 M DHP B, respectively. The binding affinity of heme appears to be significantly smaller in both isoforms of DHP than in myoglobins. This observation was corroborated by heme transfer experiments, in which heme was observed to transfer for DHP A and B to horse skeletal muscle myoglobin (HSMb). GdnHCl-induced denaturation suggests a threshold of 1 mM for stabilization by binding of the inhibitor 4-bromophenol (4-BP). Concentrations of 4-BP greater than 1 mM caused destabilization. Urea-induced denaturation showed only destabilizing effects from phenolic ligand binding. Heme transfer experiments from DHP to HSMb further support the hypothesis that the binding of halophenols to DHP facilitates the removal of the heme. Thermal denaturation assessed via UV-visible spectroscopy and that assessed by differential scanning calorimetry (DSC) are both in agreement with chemical denaturation experiments and show that the denaturing abilities of the halophenols improve with the size of the para halogen atom in 4-XP, where X = iodo, bromo, chloro, or fluoro (4-IP > 4-BP > 4-CP > 4-FP), and the number of halo substituents as in 2,4,6-tribromophenol (2,4,6-TBP > 4-BP). DHP B, which differs in five amino acids, is less stable than DHP A with ΔHcal and Tm values of 165.1 kJ/mol and 47.5 °C compared to values of 183.3 kJ/mol and 50.4 °C for DHP B and DHP A, respectively. Kinetic studies verified that DHP B has a catalytic efficiency (kcat/Km) ∼5-6 times greater than that of DHP A but showed an increased level of substrate inhibition in DHP B for both 2,4,6-TCP and 2,4,6-TBP. An inverse correlation between protein stability with respect to heme loss and catalytic efficiency is suggested on the basis of the fact that the heme in DHP B has a stability lower than that of DHP A but a catalytic efficiency higher than that of DHP A.

The application of fluoride anion as a probe for investigating the internal substrate binding has been developed and applied to dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata. By applying the fluoride titration strategy using UV-vis spectroscopy, we have studied series of halogenated phenols, other substituted phenols, halogenated indoles, and several natural amino acids that bind internally (and noncovalently) in the distal binding pocket of the heme. This approach has identified 2,4-dibromophenol (2,4-DBP) as the tightest binding substrate discovered thus far, with approximately 20-fold tighter binding affinity than that of 4-bromophenol (4-BP), a known internally binding inhibitor in DHP. Combined with resonance Raman spectroscopy, we have confirmed that competitive binding equilibria exist between fluoride anion and internally bound molecules. We have further investigated the hydrogen bonding network of the active site of DHP that stabilizes the exogenous fluoride ligand. These measurements demonstrate a general method for determination of differences in substrate binding affinity based on detection of a competitive fluoride binding equilibrium. The significance of the binding that 2,4-dibromophenol binds more tightly than any other substrate is evident when the structural and mechanistic data are taken into consideration.