Studies of the Mechanism of Phenol Hydroxylase: Mutants Tyr289Phe, Asp54Asn, and Arg281Met † Dong Xu, David P. Ballou,* and Vincent Massey* Department of Biological Chemistry, UniVersity of Michigan, Medical School, Ann Arbor, Michigan 48109-0606 ReceiVed May 10, 2001; ReVised Manuscript ReceiVed August 22, 2001 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. Aromatic compounds are the second largest organic group existing in nature, next only to the carbohydrates. They are mainly found in the plant kingdom, and derive in large measure from lignin of plants (1). With the development of modern industry, phenolic compounds are also produced as plastics, resins, dyes, etc., many of which become toxic waste after their usage. Hence, the degradation of aromatic materi- als is very important for the carbon cycle and for detoxifi- cation of waste products produced by humans. Phenol hydroxylase (PHHY, 1 EC 1.14.13.7) is a flavin-containing monooxygenase from the aerobic topsoil yeast Trichosporon cutaneum (2). It is the first enzyme of a pathway this species uses for the degradation of phenolic compounds (3). Because of this pathway, T. cutaneum is able to utilize phenol as its sole carbon and energy source. PHHY is one of the more fully characterized flavoprotein aromatic hydroxylases (4). In the reaction it catalyzes (5, 6) (Scheme 1), the flavin of phenol hydroxylase is first reduced by NADPH. After releasing NADP + , the reduced flavin reacts with oxygen to form intermediate I (C4a-hydroper- oxyflavin) that is used to hydroxylate the substrate, phenol, or its derivatives, at the ortho position. The immediate pro- duct of the hydroxylation is termed intermediate II, which undergoes further change to give intermediate III (C4a- † Financial support was received from U.S. Public Health Service (Grant GM 20877 to D.P.B. and Grant GM 11106 to V.M.). * To whom correspondence should be addressed. D.P.B.: Depart- ment of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0606; e-mail, dballou@umich.edu; phone, (734) 764-9582; fax, (734) 763-4581. V.M.: Department of Biological Chemistry, Uni- versity of Michigan, Ann Arbor, MI 48109-0606; e-mail, massey@ umich.edu; phone, (734) 764-7196; fax, (734) 763-4581. 1 Abbreviations: PHHY, phenol hydroxylase from T. cutaneum; PHBH, p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa; CDO, catechol 2,3-dioxygenase; FAD, flavin adenine dinucleotide; IPTG, isopropyl 1-thio--D-galactopyranoside; Pi, phosphate; FlHOOH, enzyme-bound flavin hydroperoxide; FlHOH, enzyme-bound flavin hydroxide; WT, wild-type; TCA, trichloroacetic acid; CT, charge transfer; 2,4-D, 2,4-dihydroxybenzoate. Scheme 1: Reaction Cycle and Intermediates of Phenol Hydroxylase (5, 6) 12369 Biochemistry 2001, 40, 12369-12378 10.1021/bi010962y CCC: $20.00 © 2001 American Chemical Society Published on Web 09/22/2001