Pre-Steady-State Kinetic Studies of the Reductive Dehalogenation Catalyzed by Tetrachlorohydroquinone Dehalogenase

Abstract

Tetrachlorohydroquinone dehalogenase catalyzes two successive reductive dehalogenation reactions in the pathway for degradation of pentachlorophenol in the soil bacterium Sphingobium chlorophenolicum. We have used pre-steady-state kinetic methods to probe both the mechanism and the rates of elementary steps in the initial stages of the reductive dehalogenation reaction. Binding of trichlorohydroquinone (TriCHQ) to the active site is followed by rapid deprotonation to form TriCHQ-2 and subsequent formation of 3,5,6-trichloro-4-hydroxycyclohexa-2,4-dienone (TriCHQ*). Further conversion of TriCHQ* to 2,6-dichlorohydroquinone (DCHQ) proceeds only in the presence of glutathione. Conversion of TriCHQ to DCHQ during the first turnover is quite rapid, occurring at about 25 s-1 when the enzyme is saturated with TriCHQ and glutathione. The rate of subsequent turnovers is limited by the rate of the thiol-disulfide exchange reaction required to regenerate the free enzyme after turnover, a reaction that is intrinsically less difficult, but is hampered by premature binding of the aromatic substrate to the active site before the catalytic cycle is completed.

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

“…This result agrees with computational calculation models of the reaction of TftD in which the hydroxylation step was proposed to be the rate-determining step for dechlorination (50). The involvement of C4a-flavin adduct species in the HadA reaction makes its mechanism unique from the mechanisms of reductive dehalogenation performed by tyrosine deiodinase from human (51, 52) and glutathione-dependent dehalogenase from S. chlorophenolicum (53).…”

A Switch between One- and Two-electron Chemistry of the Human Flavoprotein Iodotyrosine Deiodinase Is Controlled by Substrate

“…10) (2). A previous proposal based on a single two-electron transfer mechanism conceived in analogy to the thiol-dependent dechlorinases (20,55) was dismissed as soon as IYD was shown to act independently of thiols and contain no Cys (nor SeCys) residues in its active site (5,15).…”

“…Fluorescence intensities were measured with a Hitachi F-4500 fluorescence spectrophotometer and normalized to the initial fluorescence (F 0 ) obtained in the absence of substrate. Dissociation constants (K D ) were obtained from the non-linear fit (Origin 7) of fluorescence versus ligand concentration using Equation 2 (20). Reported K D values derive from the average value of three independent determinations, and their error is based on the results of least square fitting.…”

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…The mechanism employed by iodotyrosine deiodinase has not still been fully established, although recent structures of the enzyme bound to its substrate have provided some insight [51,156]. The enzyme contains an FMN cofactor which plays a key role in the catalytic process by carrying out a stepwise reduction of the substrate by means of sequential one-electron transfers and not through a single two-electron transfer as previously proposed [51,157,158].…”