A radical intermediate in the conversion of pentachlorophenol to tetrachlorohydroquinone by Sphingobium chlorophenolicum

Biochemistry. 2014 Oct 21;53(41):6539-49. doi: 10.1021/bi5010427. Epub 2014 Oct 6.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Benzoquinones / chemistry
  • Benzoquinones / metabolism*
  • Biocatalysis
  • Biotransformation
  • Catalytic Domain
  • Electron Spin Resonance Spectroscopy
  • Environmental Pollutants / chemistry
  • Environmental Pollutants / metabolism*
  • Hydrocarbons, Chlorinated / chemistry
  • Hydrocarbons, Chlorinated / metabolism*
  • Hydroquinones / chemistry
  • Hydroquinones / metabolism*
  • Kinetics
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / metabolism*
  • NADP / metabolism
  • Oxidation-Reduction
  • Pentachlorophenol / chemistry
  • Pentachlorophenol / metabolism*
  • Protein Stability
  • Quinone Reductases / chemistry
  • Quinone Reductases / metabolism*
  • Sphingomonadaceae / enzymology*

Substances

  • Bacterial Proteins
  • Benzoquinones
  • Environmental Pollutants
  • Hydrocarbons, Chlorinated
  • Hydroquinones
  • tetrachlorobenzoquinone
  • NADP
  • 2,3,5,6-tetrachlorohydroquinone
  • Pentachlorophenol
  • Mixed Function Oxygenases
  • pentachlorophenol monooxygenase
  • Quinone Reductases
  • tetrachlorobenzoquinone reductase, Sphingobium chlorophenolicum