World

Journal

of Microbiology

and Biotechnology,

9, 479-482

Interaction of dichloromethane (methylene chloride) with the nitrous oxide reductase from Wolinella succinogenes Chunqing Zhang and Thomas C. Hollocher* Nitrous oxide reductase from Wolinefla succinogenes was tested for benzyl viologen cation (BV+)-chlorinated methane oxidoreductase activity, using di-, tri- and tetra-chloromethanes, and for the inhibition of BV+-N,O oxidoreductase activity by these chloromethanes. No BV+-chlorinated methane oxidoreductase activity was detected. Any such activity, if it exists, must be less than 0.1% of the BV+-N,O oxidoreductase activity of the enzyme. Inhibition of the BV+-N,O oxidoreductase activity by dichloromethane was detected and was apparently reversible and non-competitive, as is the case with the small metal-ligand type inhibitors of the enzyme (e.g. acetylene, azide, cyanide and carbon monoxide). Trichloromethane was a weaker inhibitor and inhibition was not detected with tetrachloromethane. Key words: Benzyl viologen, dichloromethane, trichloromethane, W&Ala succinogenes.

inhibitor,

kinetics, nitrous

Because of certain chemical analogies between nitrous oxide (N,O) and halogenated methanes, it occurred to us that the copper-containing nitrous oxide reductase of denitrifying and related bacteria (Kroneck & Zumft 1990; Zumft & Kroneck 1990) might be able to dehalogenate halogenated methanes reductively and thereby contribute to the ability of bacteria in anoxic environments to dehalogenate these compounds (Giilli & McCarty 1989; Egli et al. 1990; Chaudhry & Chapalamandugu 1991; Mohn & Tiedje 1992). Both N,O and halogenated methanes are inert toward many kinds of reducing agents but yield to super-reduced states of transition metals (Banks et al. 1968; Meyerstein & Mulac 1968; Blackburn et al. 1977; Krone et al. 1989a, b), co-ordinate weakly or not at all with metal complexes, have comparatively high EL values (see Methods), are of similar van der Waals dimensions (e.g. 0.52 x 0.34 x 0.34 nm and 0.64 x 0.44 x 0.38 nm for N,O and dichloromethane, respectively), and are reduced dissociatively to yield N, and O- (or OH) radical on the one hand (Matheson & Dorfman 1976) and Cl- and the corresponding hydrodehalomethane (or dihalocarbene) on the other (Krone et al. 1991;

C. Zhang and T.C. Hollocher are with the Department of Biochemistry, Brandeis University, Waltham MA 02264, USA; fax: 617 736 2349. ‘Corresponding author. @ 1993 Rapid

Communications

of Oxford

oxide, nitrous

oxide reductase, tetrachloromethane,

Helvenston & Castro 1992). In addition, certain halogenated methanes (e.g. di- and tri-chloromethane) possess a dipole moment, as does N,O. Two enzymes, in addition to nitrous oxide reductase, are known to reduce N,O catalytically: nitrogenase (Hardy & Knight 1968) and carbonmonoxide dehydrogenase (Lu & Ragsdale 1991), which depend, respectively, on the strongly reduced states of MO-Fe and Ni-Fe centres. Another enzyme, the cobalamin-dependent methionine synthase, is inactivated by N,O (Frasca et al. 1986; Banerjee & Matthews 1990), presumably as the result of the oxidation of the Co(I) state of the corrinoid cofactor by N,O. The Co(I) state of corrinoids (Krone et al. 1989a, 1991; Holliger et al. 1992a), the Ni(I) state of the coenzyme F-430 component of the methyl-CoM reductase of methanogenic bacteria (Krone et al. 1989b; Helvenston & Castro 1992; Holliger et al. 1992a, b) and the putative Ni(I) state of carbonmonoxide dehydrogenase (Jablonski & Ferry 1992) can reductively dehalogenate organic compounds. Ferrohemes and reduced cytochrome P-450 monooxygenases have also been shown to reductively dehalogenate certain organic compounds, including halomethanes (Macdonald 1983; Castro et al. 1985; Mohn & Tiedje 1992). There is clearly some overlap among enzymes and cofactors that can reduce N,O or halogenated compounds, and it was of interest to test

Ltd World Journal of Microbiology and Biohchnology, Vol 9.

1993

479

C. Zhang and T.C. Hollocher nitrous oxide reductase for possible activity toward these two different but chemically analogous kinds of substrate.

Materials

and Methods

The measurement of BV+-N,O oxidoreductase activity [EL of BV+/BV’+ = -0.36 V (Michaelis & Hill 1933)] and attempted detection of BV+-chlorinated methane oxidoreductase activities were by the spectrophotometric method of Kristjansson & Hollocher (1980) under anaerobic conditions at 20 to 25°C. Dithionite was used to generate 80 to 100 jiM BV+ (& nm= 0.8 to 1.0) from 200 pM oxidized BV (Zhang et al. 1992). The N,O concentration was routinely 0 or 25 mM in the presence or absence, respectively, of a chlorinated methane, but was varied from 25 pM to 25 mM in experiments where a chlorinated methane was tested for action as an inhibitor. In most experiments, the amount of neat tri- or tetra-chloromethane added to an optical cuvette was about 10% in excess of that calculated to provide a saturated solution in water (62 and 5.2 mu, respectively). The K, for BV+ (K,,,sv) was estimated from kinetic progress curves (Kristjansson & Hollocher 1980; Teraguchi & Hollocher 1989) under conditions where BV+ was reaction limiting. Similarly, K, for N,O (hIo) was estimated using limiting amounts of N,O (generally 25 PM). Nitrous oxide reductase was purified to a purity of about 90% from W&&u succinogenes grown on formate/N,O, as described by Teraguchi & Hollocher (1989). The enzyme from this organism was selected because of its high specific activity, great stability under air and ease of purification (Teraguchi & Hollocher 1989; Zhang et al. 1991). It is unique among nitrous oxide reductases in that

it contains

a cytochrome

c domain

in addition

to

the

Cu

catalytic centre. Eb at 25°C for N,O and di-, tri- and tetra-chloromethane in aqueous solution were calculated for dissociative Z-electron reduction (e.g. CHCl, + H, + CH,Cl, + HCl) from standard free energies of formation (Weast 197.3). Standard states were: H, and N,O, 1 atm; HCl, I M in aqueous solution; other reactants/ products, neat liquid or saturated aqueous solution. The values calculated were 1.35, 0.51, 0.44 and 0.46 V, respectively, for N,O, tetra-, hi- and di-chloromethane.

Results Figure 1 shows kinetic progress curves for experiments in which 110 mM dichloromethane was tested as a substrate analogue of N,O. In the control system containing only BV+ and N,O, injection of enzyme led to rapid bleaching of BV+ as it was converted to BV’+ (Experiment A). In systems containing dichloromethane but not N,O (Experiment C), injection of the same amount of enzyme resulted in no loss of BV+ other than that due to dilution and inadvertent oxygen contamination. Thus the progress curve of Experiment C is in this regard identical to that of Experiment B, the control lacking both dichloromethane and N,O. Subsequent injection of N,O dissolved in buffer allowed BV+ oxidation to occur, but at a rate in Experiment C smaller than that observed in the absence of dichloromethane in Experiments A and B. Experiments of this

480

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Vol 9, 1993

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ENZYME

1

ENZ.

NzO

ENZ.

NzO

1 I

6oo=o.2 1

A

B

C

++

2 MlN

Figure 1. Kinetic progress curves showing that dichloromethane is an inhibitor but not an oxidizing substrate for nitrous oxide reductase from W. succinogenes. A,, decreases from top to bottom and time increases from left to right. The exhaustion of BV+ is marked by a change in slope from a finite value to zero at the bottom of the figure. (A) Control experiment in which enzyme (1.3~9) was injected at the arrow into a mixture containing BV+ (about 90 P(M) and N,O (25 mM). Oxidation of BV+ to BV2+ resulted in a rapid decrease in A,, and ultimate exhaustion of BV+. (B) Second control experiment in which the same amount of enzyme as in A and N,O were injected at their respective arrows. The final N,O concentration was 2 mrv. The BV’+-containing solution had been sparged with N, before reducing it with dithionite. (C) Same as B, except the mixture contained 110 mM dichloromethane. Note that the decrease in A, was less rapid than in Experiments A and B.

kind, with incubation times as long as 20 min prior to injection of N,O, were carried out with tetrachloromethane and trichloromethane at their saturation concentrations of 5.2 and 62 mM, respectively, and with dichloromethane between 30 and 110 mM. The enzyme exhibited no evidence of BV+-chlorinated methane oxidoreductase activity with these three compounds. Any such activity, if it exists, must be

Interaction of dichloromethane (methylene chloride) with the nitrous oxide reductase from Wolinella succinogenes.

Nitrous oxide reductase from Wolinella succinogenes was tested for benzyl viologen cation (BV(+))-chlorinated methane oxidoreductase activity, using d...
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