ma
Blotechnol. hog. 1093, 9, 608-614
Dehalogenation by Cytochrome P-450,am: Effect of Oxygen Level on the Decomposition of 1,2-Dibromo-3-chloropropane Gary S. Koe and Vincent L. Vilker' Department of Chemical Engineering, University of California at Los Angeles, Los Angeles, California 90024-1592
A P-450, monooxygenase enzyme system, obtained through separate purification of the three protein subunits, was used as a biocatalyst in the dehalogenation of 1,2dibromo-3-chloropropane (DBCP). Incubations in a solution containing the reconstituted enzyme system were performed in two oxygen environments, oxygen-rich (oxygensaturated solution) and oxygen-lean (oxygen concentration reduced to 1'?& of saturation), and analyzed for hydrocarbon and bromide ion ( B r ) product formation and the kinetics of DBCP decomposition. 1-Bromo-3-chloroacetone (BCA) was identified by gas chromatography-mass spectrometry as a major product of oxygen-rich incubations, while 3-chloro-1-propene (allyl chloride) was verified as an oxygen-lean product. The identification of these two products suggests that P-450, activity is occurring in two separate mechanisms, oxidative and reductive, which is governed by the availability of oxygen. This suggestion is further supported by differences that were observed in the DBCP decomposition rate, which is an order of magnitude slower under oxygen-lean conditions, and differences in bromide ion formation, where approximately one B r stoichiometric equivalent is released per mole of DBCP lost under oxygen-rich conditions versus approximately two B r stoichiometric equivalents for oxygen-lean conditions.
Introduction Oxygenase enzymes are being vigorously pursued for use in biocatalytic processes to remove halocarbons from water at trace concentrations of less than 0.1% (w/v). Dehalogenation of halocarbons by Pseudomonas putida PpG786 has been linked to monooxygenase activity (Castro et al., 1985, 1988; Vilker and Khan, 1989). Cloning the toluene monooxygenase gene into Escherichia coli transfers the activity to decompose trichloroethene (TCE) (Winter et al., 1989). Toluene dioxygenase in Pseudomonas putida, methane monooxygenase in methanotrophs, and ammonia monooxygenase in Nitrosomonas europaea are also implicated in trichloroethene oxidation (Wackett et al., 1989). Among this variety of oxygenases, the cytochrome P-450,- monooxygenase, with its apparent wide-ranging substrate specificity, shows much promise for carrying out bio-dehalogenation in water treatment applications. In order to utilize this system in an economically feasible process, it is essential that dehalogenation pathways, rates, and product distributions be fully characterizedas functions of substrate concentrationsand environmental conditions. Oxygen tension has been identified as one of the principal variables which must be addressed (C&E News Staff, 1991). The P-450,- monooxygenase system consists of three proteins, two of which shuttle two electrons to a terminal enzyme that catalyzes substrate (halocarbon) decomposition. Figure 1shows the established electron transport pathway when this system acts as a hydroxylase. NADH supplies the reducing power to the first subunit, putidaredoxin reductase (PdR), which in turn reduces putidaredoxin (Pd), and finally the cytochrome P-450 monooxygenase (Cyt-m) (EC 1.14.15.1). The hydroxylation cycle can be short-circuited by autodecay to superoxide and oxidized P-45Osubstrate complex or to hydrogen
* Author to whom inquiries should be addressed. 8756-7938/93/3009-0608$04.00/0
peroxide following incorporation of the second electron (Raag and Poulos, 1991). A respiring cell is capable of using the P-450, to run detoxification reactions either oxidatively or reductively (Castro and Belser, 1990,White and Coon, 1980, Guengerich and MacDonald, 1984). The key parameter is the solution oxygen tension, although the extent and details of ita influence on product distribution, kinetics, and stoichiometry are unknown. In order to better elucidate the role of oxygen in P-4500 detoxification reactions, our present research examines the decomposition of halocarbons, specifically 1,2-dibromo-3-chloropropane (DBCP),in aqueous solutions of the reconstituted enzyme system. Though realistic wastewater treatment loadings are in the sub-parts per million (ppm) range,we maintained higher substrate concentrations (ppm levels) such that substrate-to-enzyme ratios were at levels where multiple turnovers of the enzyme were assured. Substrate disappearance was simultaneouslymonitored with bromide ion release and product formation. Major products were quantified and initial rate kinetics was determined for decomposition under oxygen-rich or oxygen-lean environments.
Materials and Methods Materials. Chemicals and chromatography gels used in enzyme preparations were obtained from the following sources: bactotryptone and yeast extract, Difco Laboratories (Detroit, MI); ACS-grade mineral salts, Fisher Scientific (Fair Lawn, NJ);dithiothreitol, NADH,lysozyme, and DNase, Sigma Chemical Co. (St. Louis, MO); DEAE-Sepharose FF anion exchange gel and Sephacryl S-2OOHR size exclusion gel, Pharmacia Biotech, Inc. (Piscataway, NJ); and Affi-gel blue affinity gel, Bio-Rad Laboratories (Hercules,CA). Purified water for all media and experiments was obtained from a Milli-Q system with an HPLC-grade organic scavenging cartridge.
0 1993 American Chemical Society and American Institute of Chemical Engineers
609
Bbtmhnol. RO@,1993, Vol. 9, No. 6
V
= S-OH
0 I H
+ :* Autodecay 2H'
i
+
mos+ HO ,,
i 0;
+
mos
Figure 1. Catalytic cyle for substrate (camphor) hydroxylation by the P-450- monooxygenase system: (S)substrate, camphor; (m) nicotinamide adenine dinucleotide,reduced form; (NAD+) nicotinamide adenine dinucleotide, cytochromeP-450 hemoprotein; (NADH) oxidized form; (PdR) putidaredoxin reductase; (Pd) putidaredoxin; (el) first electron transfer; (ez) second electron transfer; (S-OH) hydroxylated substrate (5-exo-hydroxycamphor);(OZ*-) superoxide complex; (superscript 0 ) oxidized species; (superscript r) reduced species; (superscript 8) substrate-bound species; (subscript 02)oxygen-bound species. Adapted from Gunsalus et al. (1973).
Chemicals used in enzymatic decomposition experiments were obtained from the following sources: 1,2dibromo-3-chloropropane and 2-bromo-3-chloro-l-propene, Pfaltz and Bauer (Waterbury, CT); allyl chloride, Aldrich Chemical Co. (Milwaukee, WI); l-bromopropane, Eastman Kodak Co. (Rochester, NY); 99 mol ?6 hexane and dextrose, Fisher Scientific; and catalase and glucose oxidase, Sigma Chemical Co. Enzyme Preparation. Three DH5-a Escherichia coli clones used for producing each of the three proteins allowed separate purifications without the threat of cross-contamination by one of the other two enzymes and provided much higher yields of the purified proteins. Putidaredoxin Reductase (PdR). Cultures of the PdR clone were grown to cell densities of 25-30 g of wet cell pasW2.4 L of growth medium. Approximately 50 g of wet cell paste was subsequently lysed (freeze-thaw followed by the addition of 0.5 mg of lysozyme/g of wet cell paste), and the cell-free extract was purified using a procedure developed by Roome and Peterson (1988). Purified protein yields ranged around 40-50 mg/50 g of initial wet cell paste. Enyzme concentrations were determined by spectrophotometric analysis with a Beckman DU-65 spectrophotometer. A scan of PdR showed characteristic peaks a t 480, 454, 378, and 275 nm, with respective extinction coefficients of 8.5, 10.0,9.7, and 72 mM-' cm-l (Gunsalus and Wagner, 1978). Enzyme purity was determined by a ratio of absorbances at 454 and 275 nm. Putidaredoxin (Pa). The Pd cultures were grown to cell densities similar to those of PdR. The purification procedure, adapted from the methods used by O'Keefe et al. (19781, yielded approximately 20-25 mg/150 g of wet cell paste. The purified enzyme concentrations were determined by spectrophotometry. Extinction coefficients 10.4, 11.1, 15.6, and 22.8 mM-l cm-' corresponding to wavelengths of 455, 415, 325, and 280 nm, respectively, were obtained from Gunsalus and Wagner (1978). Enzyme purity was determined using the ratio of absorbances at 455 and 280 nm. Enzyme activities for PdR and Pd were obtained using the cytochrome c assay developed by
Table I. Characterization of Purified Enzyme Subunits putidaredoxin cytochrome reductase putidaredoxin P-450protein and source (PdR) (Pd) (Cyt-m) 20-25/150 2&30/50 yield (mg/g of 40-50/50 wet cell paste) 0.70-1.04 0.11-0.14 0.38-0.44 purity (as absorbance ratio)" activityb 15000 10000-2oooO 7-13 Absorbance ratio (nm/nm): PdR, 454/275 (100% = 0.14); Pd, 455/280 (100% = 0.44); Cyt-m,392/280(>95% = 1.4). Theactivities of PdR and Pd are given as moles of cytochrome c reduced/mg of pr0tein.s) using the methods described by Phillips and Langdon (1962). The activity of Cyt-m is given in units of moles of camphor consumed/mole of Cyto-ma during camphor hydroxylation.
*
Phillips and Langdon (1962). The activity of PdR was measured with NADH and Pd in excess, while PdR was present in limiting concentrations. The reverse is true for Pd activity measurements. Table I showsthe yield, purity, and activity measured for each enzyme. CytocbromeP-450(Cyt-m).The Cyt-m cultures were also grown to cell densities of 25-30 g of wet cell paste/2.4 L of growth medium. The purification procedure for Cyt-m was also adapted from methods used by O'Keefe et al. (1978) and yielded approximately 27 mg/50 g of wet cell paste. Cyt-m concentrations were measured spectrophotometrically (Vilker and Khan, 1989). Enzyme purity was determined by the ratio of absorbances a t 392 and 280 nm. The activity of Cyt-m was determined by measuring the rate of NADH oxidation during camphor hydroxylation. A ratio of 1:20:1 for Cyt-m/Pd/PdR was used to assure that Cyt-m activity was rate-limiting in this activity assay (Table I). Product Identification. DBCP incubation was carried out in 4-mL reaction vials containing 2 mL of P-450enzyme solution, blanketed with oxygen, and mildly stirred with a stir bar atop a Corning PC-320 stirredhot plate a t room temperature. Each purified protein solution was thawed at 2-6 OC and desalted with 20 mM potassium phosphate/ 100 mM potassium nitrate buffer (pH 7.4) through a Pharmacia C 10/20 chromatography column containing
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610
Table 11. Incubation Components for DBCP Decomposition
components cytochrome P-450 putidaredoxin putidaredoxin reductase glucosea -glucose oxidasea catalase'
NADH DBCPb buffer (pH 7.4Y
concentration in added aliquot 20-40 pM 28-69 pM 30-55 pM 1.0 M 1.0 mg/mL 75 mg/mL 0.3 M 5.2 mM
reaction concentration 2.5 pM 7.5 p M 2.5 pM 100 mM 0.1 mg/mL 0.14 mg/mL 10 mM 200 pM fill to total volume
a Oxygen scavenging system components used in oxygen-lean incubation. Stocksolution prepared by diseolvingpure DBCP liquid to saturation in water. Buffer consists of 20 mM KHzPO4 and 100 mM KN03.
G-10 Sephadex size exclusion gel (Pharmacia) to remove residual camphor and NADH. The solutions were remeasured by spectrophotometry for final protein concentration. All Components with the exception of DBCP were added to the reaction flask and thoroughly mixed. Table I1 shows the respective concentrations of each component used in the incubation procedure. The flask was blanketed with oxygen and gently stirred. At time zero, DBCP was added in the form of a saturated aqueous solution,5.2 mM, to a final concentration of 200 pM. After a 20-min incubation, the reaction solution was extracted by adding 100-pL aliquots to an extraction column containing 2 mL of 99 mol % hexane. The extraction column (height, 25 cm; i.d., 0.4 cm) was constructed from borosilicate glass. A series of 10 incubation reactions was run and extracted into the same 2-mL extract in order to increase the concentration of prospective products. Gas chromatography-electron impact mass spectrometry (electron energy, 70 eV) was performed on a Finnigan 9610 gas chromatograph (Finnigan/MAT,Sunnyvale, CA) equipped with a DB-624 capillary column (30 m X 0.32 mm) (J&W Scientific, Folsom, CA) interfaced with a Finnigan 4000 mass spectrometer, with an ion source temperature of 270 "C, an ion source pressure of 7.5 X Torr, and an emission current of 0.5 mA. The injector and detector temperatures were 225 and 250 "C, respectively. The column was held at 35 "C for 5 min following a 3-pL splitless injection and ramped at 6 "C/min to a final temperature of 160 "C. The spectral data were recorded using an Incos data system at a scan rate of 1 ddecade. In order to determine products formed under low oxygen tension, the following modificationsto the above procedure were adopted (1)the reaction volume was increased to 12 mL in a 12-mL reaction flask; (2) the DBCP concentration was increased to approximately 1mM to prevent substrate exhaustion; (3) an oxygen-lean environment was obtained by using Schlenk techniques [three successive evacuations to 20 in. Hg followed by refilling with argon (Shriver and Drezdzon, 1986)l; (4) a glucose oxidase/ catalase scavenging system was added to the aqueous solution in order to reduce the soluble oxygen to 1%of saturation; (5) the entire 12-mL reaction solution was extracted into 1mL of 99 mol % hexane; (6) approximately 40 pM 1-bromopropane(Kodak,Rochester,NY)was added to the hexane as an internal standard for possible quantitation of allyl chloride products; and (7) a HewlettPackard 5971 GC-mass spectrometer (electron impact energy, 70 eV), equipped with a DB-1 capillary column (30 m X 0.32 mm) (J&W Scientific) operated in a selected ion monitoring (SIM) mode, was used for product iden-
tification. The injector, detector, and column temperatures were maintainedat 200,250, and 30 "C, respectively. Stoichiometry and Kinetic Experiments. OxygenRich Incubations. Incubation reactions performed for the purposes of obtaining data on the stoichiometry of bromide ion release and on the kinetics of DBCP decomposition were also done at concentrations shown in Table 11. All reaction components with the exception of DBCP were added to a 12-mLreaction flask to a total volume of 12 mL. Oxygen was used to fill the void created by sampling. A bromide ion microprobe and micro-reference electrode (Lazar Research Laboratories, Los Angeles, CA) were submerged in the incubation solution, and their response was monitored by a Kipp & Zonen BD 112 chart recorder. Protein desalting, as described previously, also removed chloride ion present in the purification buffers, thereby eliminating a major interference in the bromide ion probe response. A time constant for the lag in the response of the bromide ion probe was determined by adding known amounts of sodium bromide to a reaction solution in the absence of DBCP. Bromide ion formation kinetics was adjusted using this time constant (0.020 f 0.005 min/pM B r ) . A calibration curve for bromide ion concentration as a function of probe response (millivolts) was constructed using authentic samples of sodium bromide salt solutions. Enzyme incubation reactions were initiated by adding saturated DBCP solution to an incubation mixture which contained all of the other components and in which the B r ion probe had achieved an initial stable reading. A 100-pLsample was immediately taken and extracted into 10mL of hexane. This procedure took approximately 1012 s. Samples were also extracted at reaction times of 1, 2, 5, 10, 20, 40, and 80 min. Bromide ion release was monitored simultaneously. An incubation solution with no NADH was used as the control. Gas chromatographic analysis was performed using a Varian 3300 gas chromatograph equipped with a DB-624 megabore column (30 m X 0.53 mm, J&W Scientific) and an electron capture detector (GC-ECD). The following operating conditionswere used injector temperature, 180 "C; detector temperature, 310 "C column temperature program, 50 "C for 5 min, ramp at 6 "C/min to 160 "C, hold at 160 "C for 5 min. Nitrogen (99.999% purity) was used as the carrier gas (4 mL/min) and also as a make-up gas (25 mL/min). A DBCP calibration curve was constructed using known concentrations of an authentic sample. Initial DBCP concentrations in the incubation mixtures were measured to a precision of 209 f 4 pM. Omen-LeanIncubations. The incubation procedure for the kinetics of DBCP decompositionand Brformation under oxygen-lean conditions was the same as the procedure for oxygen-rich incubations, except for these adjustments: (1) Schlenk techniques and an oxygen scavengingsystem were used to obtain the desired minimal oxygen environment; (2) argon was used to fill headspace; (3) P a r d i (American National Can, Greenwich, CT) was placed between the bromide ionheference electrode probes and the top of the reaction flask to reduce air leakage into the reaction flask; (4) sample times were extended to 1,20,30,80, and 135 min due to the slower reaction rate. Other Halocarbon Incubations. Allyl chloride and 2-bromo-3-chloro-1-propenewere each incubated under oxygen-rich conditions using the procedure for oxygenrich incubation of DBCP. Both substrates were used as the reaction initiator by the addition of a 5.2 mM aqueous solution to a final concentration of 200 pM. Unlike the DBCP solution, however, methanol was added to help
Bbtechd. Rog., 1993, Vol. 9, No. 6
m/z
Figure 2. Comparison of electron impact mass spectrum of DBCP decomposition product under oxygen-richconditions with the spectrum of l-bromo-3-chloroacetone (BCA) from Pearson et al. (1990): (A) DBCP decomposition product; (B) BCA.
dissolve the substrates (l%, v/v). The reactions were carried out while monitoring only the rate of substrate loss. Allyl chloride and 2-bromo-3-chloro-l-propene concentrations were determined at the GC-ECD conditions described above using calibration curves constructed from data on authentic samples. Control incubations were also run in the absence of NADH.
Results and Discussion Control experimentsunder oxygen-richand oxygen-lean conditions were performed to demonstrate that decomposition of DBCP was solely due to P-450,, enzyme activity. When any one component of the enzymatic pathway was omitted, DBCP decomposition or product formation was not observed. Further experiments run in the presence of metyrapone, a known P-450 inhibitor, gave similar results. Incubations containing a full complement of P-450, components reduced DBCP to less than detectable levels (5 ppb) and gave the products described below. Product Identification. Oxygen-RichIncubation. Mass spectral data of hexane-extracted reaction samples indicate that l-bromo-3-chloroacetone (BCA) is a major product of DBCP decomposition under oxygen-rich conditions. Figure 2 compares the electron impact mass spectrum to a mass spectrum obtained by Pearson et al. (1990). Abundances at mass-to-charge ratios of 170,172, and 174 are rationalized as the parent molecular ion pattern. Loss of a methyl chloride fragment gives the 1211123 pattern, while fractionation between carbons 1 and 2 gives a methyl bromide (mlz 93/95) and the
011
remaining chlorinated ion (mlz 77/79). A similar mass spectrum for BCA has also been obtained by Milano et al. (1990). An oxidative dehalogenation pathway that produces BCA is shown in Figure 3. The P-450, monooxygenase system oxidizes the carbon 2 position of DBCP, producing an unstable bromohydrin which spontaneously eliminates H + B r to form the BCA product. Omichinski et al. (1988) and Pearson et al. (1990) have also measured DBCP decomposition with whole-cell extracts containing liver microsomal P-450 enzyme. They found that, in an oxygen environment, the enzyme can hydroxylate each of the three carbons of 1,2-dibromo-3-chloropropane(DBCP) to yield a number of metabolites, including BCA if carbon 2 is hydroxylated. Omichinski et al. (1988) have also found 2-bromoacrolein as a product from the microsomal P-460 decomposition of DBCP. Formation of 2-bromoacrolein is believed to occur from the oxidation of either carbon 1 or 3 to form the corresponding unstable halohydrins, with spontaneous elimination of HBr and HC1 to give 2-bromoacrolein. No %-bromoacroleinwas found in any of our P-450, incubations with DBCP. The absence of 2-bromoacrolein from PdBO,-regulated decomposition reactions may suggest a greater specificity in the reaction pocket compared to microsomal P-450. Although a product of this nature may be produced in such minute quantities that it was undetected by our methods, it is apparently not a major product of P-450, decomposition. At present, an authentic sample of BCA is not available for quantitative analysis and rate studies. Oxygen-LeanIncubations. Retention time matching with authentic samples gave us our first indication of allyl chloride as a first generation product of reductive DBCP decomposition. An observed retention time of 3.41 & 0.05 min was in 99% agreement with the authentic sample. This determination was verified by mass spectrometry operating in a selective ion monitoring (SIM) mode. Abundances at mass-to-charge ratios 76 (molecular ion), 41 (ion without chlorine atom), and 78 (chlorine isotope) were selected for comparison. A reaction sample was then run while specificallymonitoring these peaks. The ratios 76/41 and 78/41 were compared to that of an authentic sample for compound verification. The ratios 76/41 and 78/41 obtained for SIM analysis agreed with 92.2% and 99.8 % accuracy, respectively. The formation of allyl chloride under oxygen-lean conditions supports a hypothesized mechanism for reductive dehalogenationshown in Figure 3. In the absence of oxygen, the P-450, system acts to shuttle electrons from NADH to the substrate, eliminating two bromide ions. We believe that this mechanism occurs only when the solution oxygen tension is significantly low. This is supported by the observed formation of allyl chloride in oxygen-leanincubations only. An oxygen-leanexperiment run in the absence of the glucose oxidase/catalase oxygen scavengingsystem yielded trace amounts of allyl chloride. Although we expected to see only oxidative product under these conditions, we believe that the Schlenk techniques used to reduce oxygen tension were at least significant enough to activate the reductive mechanism, yielding allyl chloride in barely detectable quantities (the allyl chloride detection limit was approximately 1pM or 70 ppb). The soluble oxygen concentration using the Schlenkpurge and refill technique was approximately 70 NM;the oxygen scavenging system further reduced the oxygen concentration to approximately 2.4 pM. The concentration of allyl chloride measured in the presence of the oxygen scavenger was approximately 10 pM; although only 5% of
612
Reduction
(+W
(+W
Oxidation (-2e)
I I
the initial DBCP concentration, it is significantly higher than the quantities measured in the absence of the oxygen scavenger. On the basis of these results, it appears that the activity of the reductiive mechanism and subsequent production of allyl chloride are significantly influenced by the availability of oxygen. Since the formation of BCA is also present in the oxygenlean incubations, we suppose that the concentration of oxygen is not so low that the process becomes strictly reductive, but rather that both reaction mechanisms are occurring simultaneously. This seems likely since our methods of lowering oxygen concentration, primarily governed by the addition of a glucose oxidase/catalase scavenger, reduced the oxygen concentration such that nontrivial quantities still remain. Kinetic and StoichiometricAnalysis. Oxygen-Rich Incubations. DBCP incubations under oxygen-rich conditions and enzyme ratios of 1:3:1 for Cyt-m/Pd/PdR were shown to decompose 90% of the substrate in a period of 5 min (Figure 4). Rate data summarized in Table I11 were evaluated for approximately 50 enzyme turnovers; a t substrate exhaustion, about 20 min, enzyme turnover was 80. The turnover rate, about 0.57 mol of DBCP consumed/mol of Cyt-ms,reveals DBCP to be aless active substrate for P-450,- relative to camphor, where a turnover rate was measured at about 10 mol of camphor consumed/mol of Cyt-ms (Table I). Although Gunsalus et al. (1973) have measured the camphor hydroxylation turnover as high as 17 s-l (corresponding to the transfer of the second electron from putidaredoxin to the terminal cytochrome), White et al. (1984) measured camphor hydroxylation a t about 1s-l when the Cybm/Pd/PdRratio was 1:4:1, which is similar to the ratio used in these experiments. This indicates a possible method of increasing DBCP reaction rates by adjustment to optimal enzyme ratios.
250
:
200
-
150
:
100
:
50
L
200
e
0
20
40
60
80
100
120
140
Time (minutes)
Figure 4. Comparison of kinetic rates of DBCP decomposition and Br- product formation for oxygen-rich and oxygen-lean incubations: (A) oxygen-rich incubation; (B) oxygen-lean incubation; ( 0 )DBCP; ( 0 )Br-.
Figure 4 also shows bromide ion formation during oxygen-rich incubation. For DBCP decomposition under these conditions, we observed that, at about 99 % DBCP
B&techrwl. Rug, 1993, Vol. 9, No. 6
Table 111. Rate Constant Comparison with Other Biocatalytic Methods sample type k-d (M-ls-l) 3900 k 560 reconstituted P-450m, oxygen-rich 268 140 reconstituted P-450-, oxygen-lean 750 Psuedomonus putida PpG786 whole cella reduced cytochrome mb 0.07 synthetic heme (PFe2+)b 0.016 a Adapted from Vilker and Khan (1989). Fractional turnover conditions, strictly reductive conditions in which the oxidation of the hemefromPFe2+toPFe3+is the measure of DBCP decomposition, from Castro et al. (1988).
615
Table IV. Relative Kinetics for Other Halocarbons
halocarbons H&--CH-CHz
I I
Br Br
I
E+S-E+P (1) -d[Sl/dt = kover~[El [SIo (2) Since extensive enzyme kinetics has yet to be determined, the overall rate constant is used here only as a means of comparison and does not imply mechanistic significance. Table I11 compares calculated overall rate constants with the rate constants from a number of other sources. P-450, containing Pseudomonas putida is capable of detoxifying DBCP under air-saturated conditions. A rate constant adapted from the work of Vilker and Khan (1989) is less than that observed in our oxygen-rich studies, suggesting that the decomposition pathway may be hindered by diffusional limitations into or within the cell or by differences between the availability of oxygen within the cell and in the reconstituted system. Finally, the measurements of heme oxidation by Castro et al. (1988) during DBCP degradation when only dithionite-reduced Cyt-m is present (NADH, PdR, and Pd are not needed for single turnover kinetic studies) give a value for k o v e r d that is significantly lower than our result under multiple turnover. Perhaps the measurements of Castro et al. are more truly reflective of the strictly reductase activity associated with this class of enzymes, rather than DBCP conversion by a faster oxidative pathway. However, we tend to think that the reason for this substantial discrepancy in rate lies with the omission of the other
k/kDBCP,oP oxygen-lean incubations" 0.07
CI
(DBCP) H&=CH-CHz
L LI
0.06
not determined
0.008
=0.00
(2-bromo-3-chloro-1 -propene) H&=CH-CH*
conversion ( ~ 8 - 1 0min), one B r ion was released for every DBCP molecule lost. This offers further support that the P-450, system is able to hydroxylate DBCP to yield BCA, as proposed by the mechanism in Figure 3. Oxygen-LeanIncubations. Under oxygen-lean incubations, the rate of DBCP decomposition was much slower: 90% conversion after about 80 min (Figure 4B). We also observed that, at 99% conversion (after more than 100 min), the stoichiometric relation of bromide ion release for each molecule of DBCP decomposed was close to 2. This suggests that the initial reaction occurring under low oxygen tension involves the release of two B r ions, which supports the hypothesized reductive dehalogenation pathway shown in Figure 3. The 10-folddecrease in the rate of DBCP decomposition under oxygen-lean conditions shows that oxygen plays a key role in the decomposition of DBCP, either by limiting the rate of the oxidative reaction pathway or by promoting a much slower reductive reaction to take place. The products formed at low oxygen suggest that both are true and that DBCP decomposition through the oxidative pathway is becoming limited due to the lack of oxygen, increasing the significance of an alternate reductive decomposition reaction. In order to facilitate the comparison of our rate determinations for DBCP decomposition with measurements by others, an overall kinetic rate constant, k o v e r d , was determined from the initial rate data assuming the following bimolecular reaction:
k/kDBcP,o, oxygen-rich incubations" 1
I
CI
(allyl chloride) a Ratio of the overallkinetic Constantsevaluated for each substrate/ the overall rate constant for DBCP decomposition under oxygenrich conditions.
P-450, subunits from the reductive, fractional turnover incubations. Pd especially is known to have more than just an electron-transfer function associated with its role in P-450, activity (Gunsalus et al., 1973). Castro et al. have also provided an overall rate constant for reductive dehalogenation using a synthetic heme, the active site of Cyt-m. Comparison of this constant in a purely global sense to that obtained for the dithionite-reduced Cyt-m and our reconstituted system suggests that the presence of the apoprotein and the remaining subunits has a significant effect on the catalytic ability of this system. A mechanistic interpretation of these results is beyond the scope of this study. We have also compared the DBCP decomposition rates to our measurements of P-450,-mediated decomposition of other low molecular weight halocarbons. Table IV shows the relative rates of decomposition for DBCP, 2-bromo3-chloro-l-propene, and allyl chloride. In an oxygen-lean environment, allyl chloride concentrations did not diminish in the presence of the enzyme system; however, we unexpectedly observed a slight decrease in allyl chloride concentration under oxygen-rich conditions compared to the controls. These observations suggest that secondary reactions may take place followingthe initial hydroxylation of DBCP under oxygen-rich conditions. This hypothesis could explain the slow increase observed in bromide ion concentration beyond one stoichiometric equivalent after DBCP is essentially exhausted in the incubation solution (see Figure 4A). In order to determine the relevance of this argument, the enzymatic decomposition of 2-bromo3-chloro-1-propene (a model dihalohydrocarbon) was investigated. We observed that the P-450- enzyme system is able to decompose 2-bromo-3-chloro-1-propene at about 10 times the rate of allyl chloride decomposition, but significantly lower than the rate of DBCP decomposition (16-17 times slower). These results suggest that a dihalohydrocarbon is susceptible to enzymatic attack, although at significantly reduced rates. Though an authentic sample of BCA is necessary to confirm the results, these experiments offer support to our observations about the extent of bromide ion release with parent DBCP substrate decomposition. BCA Cyt-m DBCP e
Notation 1-bromo-3-chloroacetone cytochrome P-450 hemoprotein 1,2-dibromo-3-chloropropane
electron transfer
Biotechnol. hog., 1993, Vol. 9, No. 8
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[El fP
%I koverd
mlz
NAD+ NADH
02.P Pd PdR
[SI [SI0 X
enzyme concentration (pM) flavoprotein (putidaredoxin reductase) percent abundance overall rate constant (L mol-' 8-1) mass-to-charge ratio &nicotinamide adenine dinucleotide (oxidized form) Bnicotinamide adenine dinucleotide (reduced form) superoxide complex product putidaredoxin putidaredoxin reductase substrate concentration (pM) initial substrate concentration (pM) halogen atom
Acknowledgment We thank Dr. Julian A. Peterson of the University of Texas Health Science Center at Dallas for providing the enzyme producing recombinant organisms and much advice on this project. We thank Ed Ruth for all work conducted on oxygen-rich product analysis. We thank Dr. Arthur K. Cho of the Department of Pharmacology at UCLA for providing access to the GC-mass spectrometer used to identify allyl chloride. All mass spectrometry work on oxygen-lean product analysis was conducted by Debbie Schmitz. We also acknowledge the fine work of Eva Escobar,Richard Looker, and Anat Shiloach. Thisproject was supported by the University of California Water Resources Center, NSF Biotechnology Grant No. EET8807418,NSF Engineering Research Center for Hazardous Substance Control, the Du Pont Company, and NIGMS/ UCLA Biotechnology Training Program. Literature Cited Castro, C. E.; Belser, N. 0. Biodehalogenation: Oxidative and reductivemetabolismof 1,1,2-trichloroethaneby Pseudomonas putida-biogeneration of vinyl chloride. Environ. Tox. Chem. 1990,9,707-714. Castro, C. E.; Wade, R. S.; Belser, N. 0. Biodehalogenation: Reaction of cytochrome P-450 with polyhalomethanes. Biochemistry 1985,24,204-210. Castro, C. E.; Yokohama, W. H.; Belser, N. 0. Biodehalogenation: Reductive reactivities of microbial and mammalian cytochromes P-450 comparedwith heme and whole-cellmodels. J. Agric. Food Chem. 1988, 36 (5), 915-919. Chemical and Engineering News Staff. Bioremediation: Innovative technology for cleaning up hazardous waste. Chem. Eng. News 1991, Aug. 16,23-44. Guengerich, F. P.; MacDonald, T. L. Chemical mechanisms of catalysis by cytochrome P-450 A unified view. Acc. Chem. Res. 1984, 17, 9-16.
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