The regulatory implications of hydroquinone for the multifunctional enzyme dehaloperoxidase-hemoglobin from Amphitrite ornata.

Hydroquinone (H2Q) has been observed to compete with the oxidation of substrates 2,4,6-tribromophenol (2,4,6-TBP) and 2,4,6-trichlorophenol (2,4,6-TCP) catalyzed by the dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata in the presence of H2O2. This competition is observed as a lag phase during which H2Q is preferentially oxidized to 1,4-benzoquinone (1,4-BQ) while totally inhibiting either 2,4,6-TBP or 2,4,6-TCP oxidation. The inhibition by H2Q is distinct from that of the native competitive inhibitor 4-bromophenol (4-BP) since H2Q is itself oxidized and its product 1,4-BQ is not an inhibitor. Thus, once H2Q is completely consumed, the inhibition is removed, and normal substrate turnover is initiated, which explains the lag phase. To probe the mechanism of lag phase, the reactions between H2Q and DHP were both studied both in the presence and in the absence of H2O2. The reversible reactions between ferric/oxyferrous DHP A and H2Q/1,4-BQ are shown to involve a proton-coupled electron transfer (PCET) mechanism, where the distal histidine His(55) serves as the proton acceptor. The pKa of the distal histidine His(55) has been determined by resonance Raman spectroscopy in order to corroborate its involvement in this mechanism. Consistent with the proposed mechanism, kinetic assays have shown that H2Q serves as a substrate for DHP that follows the Michaelis-Menten kinetics. Unlike H2Q, the product 1,4-BQ has a relatively large Ki value and therefore has negligible inhibition. This study sheds light on understanding the difference between substrate and inhibitor binding sites and regulatory implication for the peroxidase and oxygen-transporter functions in DHP. It also provides information on PCET in DHP, which is important for resolving the switching between the ferric peroxidase catalytic function and the ferrous oxygen transport function.