Environ. Sci. Technol. 1996, 30, 1752-1755
Degradation of Metal-Nitrilotriacetate Complexes by Nitrilotriacetate Monooxygenase L U Y I N G X U N , †,‡ R O B E R T B . R E E D E R , †,‡ ANDREW E. PLYMALE,‡ DONALD C. GIRVIN,§ AND H A R V E Y B O L T O N , J R . * ,‡ Department of Microbiology, Washington State University at Tri-Cities, Richland, Washington 99352, and Environmental Microbiology Group and Interfacial Geochemistry Group, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352
Studies of metal-NTA complex degradation using NTA monooxygenase (NTA-Mo) can provide a mechanistic understanding of NTA degradation and lead to approaches to remediate recalcitrant metalNTA complexes (e.g., NiNTA-). NTA can exist in aqueous systems as various species depending upon the pH and types and concentrations of ions present (e.g., HNTA2-, CaNTA-, MgNTA-). An understanding of the aqueous speciation of NTA is necessary to determine the substrate range of NTA complexes degraded by NTA-Mo. The protonated form of NTA (HNTA2-) and CaNTA- were not degraded by NTAMo, while MgNTA-, MnNTA-, CoNTA-, FeNTA-, NiNTA-, and ZnNTA- were degraded with similar Km’s. This is surprising because these metal-NTA complexes have different rates of biodegradation by whole cells. This suggests that biodegradation of various metal-NTA complexes is limited by the rate of transport into the cell and that NTA-Mo may be useful for degrading metal-NTA complexes recalcitrant to degradation by whole cells. In mixed systems containing both substrate (MgNTA-) and nonsubstrate (CaNTA-), aqueous speciation modeling was able to provide the substrate concentration, which correlated well with the rate data (r2 ) 0.95). This demonstrates that aqueous speciation modeling can be used to predict the rate of NTA degradation by NTA-Mo for complex systems containing multiple species.
* Corresponding author address Pacific Northwest National Laboratory, Mail Stop K4-06, 900 Battelle Blvd., Richland, WA 99352; telephone: (509) 375-2758; fax: (509) 375-6666; e-mail address:
[email protected]. † Washington State University at Tri-Cities. ‡ Environmental Microbiology Group. § Interfacial Geochemistry Group.
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 5, 1996
Introduction Nitrilotriacetate (NTA) is a synthetic chelating agent that has been used for various nuclear waste processing procedures (1-3) and has been co-disposed to the soils and sediments with radionuclides and heavy metals. Because chelating agents form soluble complexes with radionuclides, their presence can increase the mobility of radionuclides and metals in the subsurface environment (4, 5). As a result, microbial degradation of chelating agents like NTA may ultimately decrease the transport of radionuclides and metals in the environment. Synthetic chelating agents such as NTA can have various species present in solution (e.g., HNTA2-, MgNTA-, CaNTA-) depending upon the pH and the concentrations and types of ions present. This aqueous speciation of NTA is critical to understanding the species being degraded as well as the process of degradation. Several studies have investigated metal-NTA complex degradation with intact cells using aqueous speciation modeling to assist in identifying the dominant forms of NTA in solution (6-8). While useful, these studies do not target individual cellular mechanisms including transport into the cell, possible respeciation in the cell cytoplasm, and finally the enzymatic degradation of the NTA and/or the metal-NTA complex (7). Enzymatic studies are therefore necessary for a mechanistic understanding of chelate degradation without the confounding influence of NTA transport inside the cell cytoplasm. Also, contaminant degradation via in vitro or ex-situ enzymes has been suggested as a viable remediation strategy (9, 10). This is especially true for waste sites, which are not conducive to microbial growth or activity (e.g., extremes of pH, salinity, temperature, metal and radiation content) or if the substrate range for the enzyme is more extensive than for whole cells. The first enzyme for the degradation of NTA, nitrilotriacetate monooxygenase (NTA-Mo) from Chelatobacter heintzii (11), has been purified and studied (12). Our objective was to use NTA-Mo to investigate how the aqueous speciation of NTA influences its enzymatic degradation. This would offer insight into the possible mechanism of degradation as well as suggest remediation strategies for metal-NTA complexes that are recalcitrant to degradation by whole cells or for contaminated environments not conducive to microbial activity.
Materials and Methods Enzyme Preparation. C. heintzii ATCC 29600 (11, 13) was grown with 5.23 mM NTA and 12.2 mM acetate in a defined liquid medium (7), which resulted in HNTA2- as the dominant aqueous species (88%) with MgNTA- (9%) and CaNTA- (3%) also present (7). NTA-Mo was then partially purified by a modification of the method of Uetz et al. (12) as follows. Cells were centrifuged and suspended in pH 8 buffer (20 mM Tris, 5 mM EDTA), lysed with a French press (18000 psi), and nucleic acids were precipitated with protamine sulfate (0.5 mg/mL). Solid (NH4)2SO4 was added to 33% saturation, and stirred for 10 min, and the solution was centrifuged (17000g, 6 °C). Additional (NH4)2SO4 was added to the supernatant (65% saturation), and the solution was stirred and centrifuged as above. The pellet with NTAMo activity was dissolved in pH 8 buffer (20 mM Tris, 5 mM
0013-936X/96/0930-1752$12.00/0
1996 American Chemical Society
EDTA) and centrifuged. All subsequent protein purification steps were performed at 6 °C. The supernatant was loaded onto a phenyl agarose (Sigma) column equilibrated with pH 8 buffer [20 mM Tris, 1 mM EDTA, 1 mM dithiothreitol, 25% (NH4)2SO4]. The enzyme was eluted with a linear gradient of 20-0% (NH4)2SO4. The two components of the enzyme eluted at around 12.5% (NH4)2SO4. Solid (NH4)2SO4 was then added to 65-70%. The precipitated proteins were centrifuged (17000g, 10 °C), resuspended in pH 7.8 buffer [20 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer, 100 mM NaCl, 1 mM dithiothreitol], and dialyzed against 1 L of pH 7.8 buffer for 2 h. The partially purified, dialyzed NTA-Mo was stored at -80 °C. The enzyme was stable over a 3-month period. The protein concentration was 23.8 mg/mL as determined by the protein-dye binding assay developed by Bradford (14). The specific enzyme activity increased from 0.027 to 0.19 µmol min-1 mg-1 of protein from the cell extracts to the partially purified enzyme using MgNTA- as the substrate. This enzyme preparation had little nonspecific NADH oxidation about 0.006 µmol min-1 mg-1 of protein in the absence of NTA. This low nonspecific NADH oxidation indicates that this NTA-Mo did not contain decoupled subunits. NADH oxidation was calculated by using the molar extinction coefficient for NADH equivalent to 6200 A340 cm-1 mol-1. Enzyme Assay and Activity. The reaction mixture contained 20 mM HEPES buffer (pH 7.6), 10 µM flavin mononucleotide (FMN), 700 units/mL catalase (from bovine liver, Sigma), 95 µg/mL partially purified NTA-Mo, 5 mM NADH, and various mixtures of cations and NTA, as indicated below in a total volume of 250 µL. All reactions were incubated at 24 °C for 1.5-3 min. Degradation of NTA was determined by measuring the formation of glyoxylate. Glyoxylate concentration was monitored spectrophotometrically after reaction with phenylhydrazineK3Fe3(CN)6 (15). The baseline for glyoxylate was established using the reaction mixture without enzyme. The substrate concentration was plotted against reaction rates in double reciprocal plots to provide Km and Vmax (16). Vmax is the maximum enzymatic rate with unlimited substrate, while Km is the substrate concentration at one-half Vmax. Aqueous Speciation of NTA. The distribution of NTA among its free acid and complexed forms (e.g., CaNTA-, CoNTA-) was calculated using the aqueous speciationsolubility model MINTEQ and its associated database (17). These calculations were based on the measured concentrations of cations, anions, pH, and NTA and the thermodynamic stability constants for H3NTA and cation-NTA complexes (17-20). Whenever we discuss the dominant NTA species in solution, we are referring to the species distribution as calculated using MINTEQ. Inductively coupled plasma/atomic emission spectroscopy was used to measure the concentrations of cations in reagents and enzyme preparations.
Results and Discussion When only NTA was added at 2 mM to the assay, the dominant species was HNTA2- even in the presence of the measured concentrations of cations present. The HNTA2was not degraded by NTA-Mo. This was also reported by Uetz et al. (12). This is not too surprising even though whole cells degraded HNTA2- faster than several cationNTA complexes including CoNTA-, FeOHNTA-, ZnNTA-,
AlOHNTA-, CuNTA-, and NiNTA- (7). Presumably the transport of HNTA2- into the cytoplasm of the NTA degrader would allow the NTA to form cation-NTA complexes such as MgNTA-. Several cations were assayed to determine whether they would allow NTA-Mo to degrade NTA. A mixture of 2 mM NTA and 0.1 mM metal as the chloride salt was used for this experiment. This would result in a maximum of 5% of the NTA being present as the cation-NTA complex with the remainder as HNTA2-. The addition of cations influenced the degradation and aqueous speciation of NTA in three ways. First, the addition of Mg2+, Mn2+, Ni2+, Co2+, Zn2+, Fe3+, or Fe2+ resulted in the degradation of NTA with only a small portion of the NTA (usually ≈4-5%) present as the cation-NTA complex. The maximum amount of the metal-NTA complex degraded during this assay was 20%. The Fe3+ added to the assay mixture may have been reduced to Fe2+, resulting in Fe(II)NTA species instead of Fe(III)NTA species. Second, the addition of Cr2+, Ba2+, Sr2+, Cr3+, K+, or Na+ resulted in no degradation of NTA. The fact that NTA was present as HNTA2- could explain these results. Third, the addition of Ca2+, Cu2+, Cd2+, or Al3+ resulted in no degradation even though the cation-NTA complex (or multiple cation-NTA complexes in the case of Al) was present (usually ≈4-5%). Uetz et al. (12) previously reported that NTA-Mo in cell extracts could oxidize NTA in the presence of Mg2+, Co2+, or Mn2+, but not Ca2+ or Cu2+ as we found here. Thus, not all cationNTA complexes are substrates for NTA-Mo, and it is presently unclear why certain cation-NTA complexes do not result in NTA degradation by NTA-Mo. Uetz et al. (12) previously reported that NTA-Mo in cell extracts could not oxidize NTA in the presence of Fe2+, Fe3+, Ni2+, or Zn2+. We found that these metals did promote NTA biodegradation by NTA-Mo at lower metal concentrations (0.1 vs 1.6 mM). We then tested solutions containing equimolar Ni2+ or Zn2+ and NTA and found that NTA degradation did occur at concentrations as high as 4 mM (data not shown). In these solutions virtually all the NTA was present as the metal-NTA complex with the uncomplexed or ionic Ni2+ or Zn2+ at only a small fraction (
