Studies of the Mechanism of Phenol Hydroxylase: Mutants Tyr289Phe, Asp54Asn, and Arg281Met

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Abstract

Three residues in the active site of the flavoprotein phenol hydroxylase (PHHY) were independently changed by site-directed mutagenesis. One of the mutant forms of PHHY, Tyr289Phe, is reduced by NADPH much slower than is the wild-type enzyme, although it has a slightly higher redox potential than the wild-type enzyme. In the structure of the wild-type enzyme, residue Tyr289 is hydrogen-bonded with the FAD when the latter is at the "out" position but has no direct contact with the flavin when it is "in". The oxidative half-reaction of PHHY is not significantly affected by this mutation, contrary to the concept that Tyr289 is a critical residue in the hydroxylation reaction [Enroth, C., Neujahr, H., Schneider, G., and Lindqvist, Y. (1998) Structure 6, 605-617; Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. Tyr289 may help stabilize the FAD in the out conformation where it can be reduced by NADPH. For the Asp54Asn mutant form of PHHY, the initial step of the oxidative half-reaction is significantly slower than for the wild-type enzyme. Asp54Asn utilizes less than 20% of the reduced flavin for hydroxylating the substrate with the remainder forming H(2)O(2). Similar changes are observed when Arg281, a residue between Asp54 and the solvent, is mutated to Met. These two residues are suggested to be part of the active site environment the enzyme provides for the flavin cofactor to function optimally in the oxidative half-reaction. In the construction of the mutant forms of PHHY, it was determined that 11 of the previously reported amino acid residues in the sequence of PHHY were incorrect.

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