title: A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum.
abstract: Pentachlorophenol (PCP) hydroxylase, the first enzyme in the pathway for degradation of PCP in Sphingobium chlorophenolicum, is an unusually slow flavin-dependent monooxygenase (k(cat) = 0.02 s⁻¹) that converts PCP to a highly reactive product, tetrachlorobenzoquinone (TCBQ). Using stopped-flow spectroscopy, we have shown that the steps up to and including formation of TCBQ are rapid (5-30 s⁻¹). Before products can be released from the active site, the strongly oxidizing TCBQ abstracts an electron from a donor at the active site, possibly a cysteine residue, resulting in an off-pathway diradical state that only slowly reverts to an intermediate capable of completing the catalytic cycle. TCBQ reductase, the second enzyme in the PCP degradation pathway, rescues this nonproductive complex via two fast sequential one-electron transfers. These studies demonstrate how adoption of an ancestral catalytic strategy for conversion of a substrate with different steric and electronic properties can lead to subtle yet (literally) radical changes in enzymatic reaction mechanisms.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology and the Cooperative Institute for Research in Environmental Sciences, and ‡Department of Chemistry and Biochemistry, University of Colorado Boulder , Boulder, Colorado 80309, United States.
authors.forename: Johannes
authors.initials: J
authors.lastname: Rudolph
authors.forename: Annette H
authors.initials: AH
authors.lastname: Erbse
authors.forename: Linda S
authors.initials: LS
authors.lastname: Behlen
authors.forename: Shelley D
authors.initials: SD
authors.lastname: Copley
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D001711:Biotransformation; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D004785:Environmental Pollutants / Q000737:chemistry / Q000378:metabolism*; D006843:Hydrocarbons, Chlorinated / Q000737:chemistry / Q000378:metabolism*; D006873:Hydroquinones / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010416:Pentachlorophenol / Q000737:chemistry / Q000378:metabolism*; D055550:Protein Stability; D011808:Quinone Reductases / Q000737:chemistry / Q000378:metabolism*; D042301:Sphingomonadaceae / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D016227:Benzoquinones; D004785:Environmental Pollutants; D006843:Hydrocarbons, Chlorinated; D006873:Hydroquinones; C000591600:tetrachlorobenzoquinone; D009249:NADP; C044578:2,3,5,6-tetrachlorohydroquinone; D010416:Pentachlorophenol; D006899:Mixed Function Oxygenases; C069781:pentachlorophenol monooxygenase; D011808:Quinone Reductases; C000594018:tetrachlorobenzoquinone reductase, Sphingobium chlorophenolicum