Eur. J. Biochem. 267, 2498±2504 (2000) q FEBS 2000
Methyl-coenzyme M formation in methanogenic archaea Involvement of zinc in coenzyme M activation Karin Sauer and Rudolf K. Thauer Max-Planck-Institut fuÈr terrestrische Mikrobiologie and Laboratorium fuÈr Mikrobiologie des Fachbereichs Biologie der Philipps-UniversitaÈt, Marburg, Germany
Methyl-coenzyme M (2-methylthioethane sulfonate) is the key intermediate of methane formation in methanogenic archaea. It is generated from coenzyme M (2-mercaptoethane sulfonate) in methyl transfer reactions catalyzed by proteins containing zinc. Here, we report that, for methyltransferase MtaA from Methanosarcina barkeri, the zinc is involved in coenzyme M activation. For the experiments an inactive MtaA apoprotein was obtained by heterologous overproduction in Escherichia coli grown in the presence of 2 mm EDTA. The apoprotein was found to react with zinc or cobalt to the fully active holoenzyme. Appoximately 1 mol of transition metal was bound per mol of protein. Upon incubation of the holoenzyme with coenzyme M < 1 mol of proton was released per mol of zinc or cobalt. Protons were not released upon incubation of the apoprotein with coenzyme M or of the holoprotein with other thiol compounds or with methyl-coenzyme M. The findings are interpreted as indicating that the role of the transition metal in MtaA is to lower the microscopic pKa of the thiol group of coenzyme M by coordination to the zinc, and thus to increase its nucleophilicity for methyl group attack. The pKZn21 of MtaA was re-determined and found to be . 15 and not 9.6 as previously reported by us. Keywords: methanogenesis from methanol; methanogenic archaea; Methanosarcina barkeri; methyltransferase; thiol group methylation.
Methyl-coenzyme M (2-methylthioethanesulfonate) is an intermediate in the energy metabolism of methanogenic archaea [1,2]. The methylthioether is generated from coenzyme M (2-mercaptoethanesulfonate) in a reaction in which the proton of the thiol group of coenzyme M is replaced by a methyl group derived from a methylated corrinoid protein [3]. The nucleophilic substitution is catalyzed by methyltransferases which can also use free methylcob(III)alamin as the methyl donor for coenzyme M methylation. CH3 2 cob
IIIalamin 1 HS 2 CoMNcob
Ialamin 1CH3 ÿ S 2 CoM
1 DG8 0 220:5 kJ´mol21 See also [4]. Under mildly alkaline conditions methyl transfer from methylcobolamin to thiols proceeds spontaneously, the pH dependence being consistent with with a nucleophilic Correspondence to: R. K. Thauer, Max-Planck-Institut fuÈr terrestrische Mikrobiologie, Karl-von-Frisch-Straûe, D-35043 Marburg, Germany. Fax: 1 49 642 117 8209, Tel.: 1 49 642 117 8200, E-mail:
[email protected] Abbreviations: BHMT, betaine:homocysteine S-methyltransferase; CH3-S-CoM, methyl-coenzyme M; H-S-CoM, coenzyme M; IPTG, isopropyl thio-b-d-galactoside; MetE, cobalamin-independent methionine synthase; MetH, cobalamin-dependent methionine synthase; Mta, methanol:coenzyme M methyltransferase; YagD, S-methylmethionine: homocysteine methyltransferase. Enzyme: methanol:coenzyme M methyltransferase (EC 2.1.1.90 and 2.1.1.-). (Received 10 January 2000, accepted 23 February 2000)
displacement of the methyl group by a thiolate anion, resulting in the heterolytic cleavage of the carbon±cobalt bond [5,6]. The methylcob(III)alamin:coenzyme M methyltransferase best studied is the one involved in methanogenesis from methanol in Methanosarcina barkeri [7]. The methyltransferase designated MtaA together with the proteins MtaB and MtaC mediate the formation of methyl-coenzyme M from methanol and coenzyme M [8,9]. MtaABC
CH3 OH 1 H-S-CoM ! CH3 -S-CoM 1 H2 O
2
DG 0 227:5 kJ´mol21 MtaC is a 28-kDa corrinoid protein, MtaB a 50-kDa protein catalyzing the methylation of MtaC and MtaA a 36-kDa protein catalyzing the demethylation of methylated MtaC. MtaB
CH3 OH 1 MtaC N CH3 ±MtaC 1 H2 O MtaA
CH3 ±MtaC 1 H±S±CoM N MtaC 1 CH3 ±S±CoM
3
4
MtaC and MtaB form a tight complex and the encoding genes form a transcription unit [9], whereas MtaA purifies separately and its encoding gene is located separately [8]. There is evidence, however, that during catalysis of reaction 2, the three subunits form a complex and interact cooperatively [10]. MtaA has been shown to contain 1 mol of zinc per mol [4,11,12]. Here we provide evidence that the role of zinc in MtaA is identical to that of zinc in the cobalamin-dependent methionine synthase MetH [13,14] which catalyzes the formation of methionine from methylcob(III)alamin and homocysteine, a reaction analogous to that catalyzed by MtaA.
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Involvement of zinc in coenzyme M methylation (Eur. J. Biochem. 267) 2499
M AT E R I A L S A N D M E T H O D S Methylcob(III)alamin, mercaptoethanesulfonate (coenzyme M) and methylmethanethiosulfonate were from Sigma. Phenol red and 4-(2-pyridylazo)resorcinol were from Fluka. Escherichia coli M15 and the expression vector pQE 30 were from Qiagen. pUH28, which is pQE30 carrying the mtaA gene, was from Harms & Thauer [8]. Heterologous overproduction of MtaA For overproduction of MtaA with an N-terminal His6-tag, E. coli M15 was transformed with pUH28. The transformed organism was grown anaerobically at 37 8C in 500 mL Luria± Bertani medium containing ampicilin (100 mg´mL21) and canamycin (50 mg´mL21) to an absorbance difference of 0.9 at 578 nm and subsequently induced by the addition of isopropyl thio-b-d-galactoside (IPTG) (0.1 mm). Two hours after induction, during which time the absorbance difference increased to < 2.0, the cells were harvested. Approximately 1 g cells (wet mass) were obtained per 0.5 L of culture. Where indicated the medium was supplemented with EDTA. In the presence of 2 mm EDTA growth was somewhat slower and also the maximal cell concentration reached was lower (DA578 , 1.5). Purification of MtaA The cells of E. coli (pUH28) (1 g wet mass) were suspended in 8 mL anaerobic 50 mm Mops/KOH pH 7.0 and the suspension was subjected anaerobically to ultrasonication at 100 W for 5 min at 4 8C. The lysate was subsequently centrifuged for 20 min at 120 000 g at 4 8C. The supernatant with 12 mg protein per mL contained all of the overproduced MtaA which was purified by FPLC on Source 15Q (XK16/10). The column was eluted by a step gradient of ammonium chloride in 50 mm Mops/KOH pH 7.0; 20 mL 0 mm; 60 mL 50 mm; 80 mL 100 mm. MtaA eluted after 40 mL 100 mm ammonium chloride. Via this procedure an over 95% pure MtaA preparation (< 10 mg) was obtained as revealed by SDS/ PAGE. Purification via this procedure was much more rapid and resulted in much higher MtaA yields than the one described previously employing chromatography of the His-tagged MtaA on Ni-nitrilotriacetic acid agarose and on Mono Q [8]. Protein was determined using the method described by Bradford [15] with Roti Nanoquant Reagent from Roth and BSA as standard. Determination of MtaA activity Assays of coenzyme M-dependent demethylation of methylcob (III)alamin were performed anaerobically in 1.5-mL cuvettes under an atmosphere of 95% N2/5% H2 at 37 8C in the dark. The 1-mL assay mixture contained: 50 mm Mops/KOH pH 7.0; 50 mm methylcob(III)alamin; 0.5 mm coenzyme M and 10±100 mU MtaA. The reaction was started by the addition of protein or coenzyme M and followed by measuring the disappearance of methylcob(III)alamin photometrically at 520 nm (D: 6.3 mm21´cm21). 1 U 1 mmol methylcob(III)alamin per min. Determination of proton release The method employed is prinicipally that described by Goulding & Matthews [13] for cobalamin-dependent
methionine synthase. The 1-mL assay mixture at 20 8C contained: 0.1 mm Mops/KOH pH 7.7; 20 mm MgCl2; 40 mm phenol red; and 5±15 mm MtaA (0.18±0.54 mg protein´mL21). The reaction was started by the addition of 0±50 mL 1 mm coenzyme M in 0.1 mm Mops/KOH pH 7.7, 20 mm MgCl2 and 40 mm phenol red. Proton release was followed photometrically at 558 nm. The change in absorbance per mol protons was calibrated by the addition of 0±30 mL 1 mm HCl (tritrisol). The MtaA used in these experiments was washed three times in Centricon 10-kDa tubes with 2 mL 0.1 mm Mops/KOH pH 7.7 containing 20 mm MgCl2. Determination of zinc Samples of MtaA (0±0.2 mg protein) were added to the 1-mL assay mixture containing 100 mm 4-(2-pyridylazo)resorcinol, 150 mm methylmethanethiosulfonate and 0.2% SDS in 50 mm Mops/KOH pH 7.0. After heating for 10 min in a boiling water bath, the assay mixture was cooled to room temperature and the absorbance at 500 nm was measured photometrically [11,16]. With ZnCl2 standards an :500 60 mm21´cm21 was determined. Determination of cobalt Cobalt was determined by atomic absorption spectroscopy as described previously [17].
R E S U LT S The aim of the experiments was to correlate the catalytic properties of MtaA with its zinc content. For this, conditions had to be found for the preparation of apoprotein, which can be re-converted to the fully active enzyme by incubation in the presence of zinc. In a previous study on the role of zinc in MtaA the apoprotein was obtained by incubation of the holoprotein in the presence of EDTA [4]. Under the conditions employed more than 99% of the enzyme was irreversibly inactivated [4]. In the meantime, we have found that native apoprotein can be obtained by heterologous expression of the mtaA gene in E. coli growing in the presence of 2 mm EDTA. The following experiments were therefore performed with MtaA apoprotein and holoprotein heterologously overproduced in E. coli. First the preparation of the apoprotein and of the holoprotein and the activity and zinc content of the preparations are described. Using these preparations, the involvement of zinc in coenzyme M activation was then investigated. Finally, the dissociation constant Kd of the zinc MtaA complex was estimated. To interpret the results it is important to know that all the solutions used in the experiments contained some contaminating zinc (. 1 mm) because it is almost impossible to obtain zinc-free solutions. MtaA apoprotein formation E. coli transformed with pQE30 carrying the mtaA gene was grown in Luria±Bertani medium supplemented with EDTA. It was found that the MtaA protein was overproduced in similar amounts at all EDTA concentrations tested as revealed by SDS/PAGE of the E. coli cell-extract proteins (Fig. 1A). The specific MtaA activity decreased, however, with increasing EDTA concentrations in the growth medium (Fig. 1B) indicating that during growth in the presence of EDTA, in addition to the holoprotein, the MtaA apoprotein was
2500 K. Sauer and R. K. Thauer (Eur. J. Biochem. 267)
q FEBS 2000
Fig. 1. Heterologous overproduction of MtaA in E. coli M15 (pUH28) grown in Luria±Bertani medium containing 0, 0.5, 1 and 2 mm EDTA. (A) SDS/PAGE of cell extract (20 mg protein) showing that EDTA had no effect on the amount of MtaA overpoduced (15% polyacrylamide; protein stained with Coomassie Brilliant Blue); (B) plot showing that the specific MtaA activity in cell extract decreased with increasing EDTA concentrations in the growth medium. E. coli was grown, MtaA synthesis induced by IPTG and MtaA activity assayed as described in Materials and methods.
also formed. This was substantiated by the finding that the specific MtaA activity of extracts from 2 mm EDTA grown cells increased from 0.02 U´mg21 to 0.2 U´mg21 when the cell extracts were supplemented with zinc and then incubated for 24 h (Table 1). Zinc content and specific activity of purified MtaA apoprotein and holoprotein MtaA purified from cells grown in the presence of 2 mm EDTA contained 0.1±0.2 mol zinc per mol and had a specific activity of 0.2 U´mg protein21; whereas MtaA purified from cells grown in the absence of EDTA contained < 1 mol zinc per mol and had a specific activity of 2.0 U´mg21 (Table 1). The zinc content thus correlated with the specific activity. MtaA purified from zinc supplement extracts of 2 mm EDTA grown cells contained < 1 mol zinc and had a specific activity of < 2.0 U´mg21 (Table 1). MtaA purified from cobalt-supplemented extracts of 2 mm EDTA grown cells contained 0.4 mol zinc and 0.6 mol cobalt per mol and had a specific activity of < 1.9 U´mg21 (result not shown). MtaA purified from M. barkeri has a zinc content of 1 mol´mol21 [11] and exhibits almost the same specific activity of 2 U´mg21 as the heterologously produced enzyme when assayed under the same conditions. MtaA activity increases linearily with the methylcob(III)alamin concentration in the range that is testable experimentally [4,10,11]. This has to be considered when comparing results. MtaA holoprotein formation from purified MtaA apoprotein and zinc Upon incubation of purified MtaA apoprotein in the presence of zinc the specific activity increased from 0.2 U´mg21 to
Fig. 2. MtaA activity increase during incubation of the purified apoprotein at 4 8C and pH 7.0 in the presence of Zn21. MtaA protein was purified from E. coli grown in the presence of 2 mm EDTA in Luria± Bertani medium. It contained 0.2 mol zinc per mol. The purified protein (0.3 mg) in 1 mL Mops/KOH pH 7.0 was incubated at 4 8C in the absence or presence of added ZnCl2 (20 mm). At the times indicated 100-mL samples were withdrawn and assayed for MtaA activity as described in Materials and methods.
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Involvement of zinc in coenzyme M methylation (Eur. J. Biochem. 267) 2501
Table 1. MtaA activity of cell extracts and of purified MtaA from M. barkeri cells grown in the absence or presence of 2 mm EDTA. For the purified MtaA protein also the Zn21 content is given. ND, not determined.
Fraction
Specific activity (U´mg21)
Extracts from cells grown in the absence of EDTA Extracts from cells grown in the presence of 2 mm EDTA MtaA purified from cells grown in the absence of EDTA MtaA purified from cells grown in the presence of 2 mm EDTA
0.2 0.02 2.0 0.2
Zn content (mol´mol21)
Specific activity after 24 h incubation with Zn21 (U´mg21)
Zn21 content after 24 h incubation with Zn21 (mol´mol21)
ND ND 1.0 0.1±0.2
0.2 0.2 2.0a 2.0a
ND ND 1.0a 1.0a
21
a
The cell extract rather than purified MtaA was incubated for 24 h at 4 8C in the presence of 0.6 mm ZnCl2 and then the MtaA protein was purified and the specific activity and zinc content determined.
1.4 U´mg21 within several hours (Fig. 2). Zn21 could be replaced by Co21 but not by Mg21, the kinetics of activation by Co21 being similarily slow (results not shown). In the absence of added Zn21 or Co21 the specific activity also increased somewhat but much more slowly (Fig. 2) probably because of the presence of contaminating zinc in the buffers. The results show that Zn21 or Co21 are required for MtaA activity. The specific activity reached after incubation of the purified MtaA apoprotein in the presence of zinc was < 25% lower (1.4 U´mg21; Fig. 2) than that of the purified holoprotein generated from the apoprotein in cell extracts (2.0 U´mg21; Table 1). Apparently, reconstitution was more complete in cell extracts. Involvement of zinc in coenzyme M activation When coenzyme M was added to MtaA at pH 7.7 the pH of the solution decreased indicating the release of protons upon
binding of coenzyme M to the protein (Fig. 3A). At saturating coenzyme M concentrations < 0.8 mol of protons were released per mol of MtaA holoprotein or per mol of Zn21 and Co21 bound to MtaA (Fig. 3B). In case of MtaA preparations with a zinc content of 0.2 mol´mol21 less than 0.2 mol protons were found per mol apoprotein (Fig. 3A,B, D). The observed release of protons suggests that coenzyme M binds with its thiol group (pKa 9.1) [18] to the zinc in the active site of MtaA forming a coenzyme M thiolate zinc complex. H±S±CoM 1 Zn±MtaANCoM±S±Zn±MtaA2 1 H1
5
Proton release was only observed when coenzyme M, rather than other thiol-containing compounds or methyl-coenzyme M, were added to MtaA. No protons were released when coenzyme M was added to MtaB, a zinc protein which activates methanol rather than coenzyme M [4]. Also no protons were released upon addition of methanol to the
Fig. 3. Release of protons upon incubation of the MtaA holoprotein with coenzyme M. (A) Coenzyme M concentration dependence at three fixed concentrations of MtaA; B, 10.7 mm holoprotein containing 1 mol Zn21 per mol; O, 7.8 mm holoprotein containing 1 mol Zn21 per mol; and K, 6.2 mm MtaA apoprotein containing 0.2 mol Zn21 per mol. (B) MtaA concentration dependence at saturating coenzyme M concentrations (50 mm); X, holoprotein containing 1 mol Zn21 per mol (11.5 mm, 10.5 mm, 7.8 mm and 5.8 mm); A, holoprotein (8.2 mm) containing 0.6 mol Co21 and 0.4 mol Zn21 per mol; K, apoprotein (6.2 mm) containing 0.2 mol Zn21 per mol. Proton release was assayed as described in Materials and methods.
2502 K. Sauer and R. K. Thauer (Eur. J. Biochem. 267)
q FEBS 2000
Fig. 4. Effect of zinc complexing agents on the activity of MtaA at pH 7.0 and 20 8C. (A) Time-dependent decrease of the MtaA activity during incubation of the holoprotein in the presence of 1 mm EDTA (O), 10 mm EDTA (B), 1 mm nitrilotriacetic acid (P) or 1 mm EDTA plus 0.1 mm ZnCl2 (1). In one of the experiments the holoprotein was first incubated in the presence of 1 mm EDTA for 100 min (O) and the incubation mixture was then supplemented with 0.1 mm ZnCl2 (K). (B) MtaA activity after 60 min incuabtion of the holoprotein in the presence of increasing concentrations of NaHS/ H2S or EDTA. Loss of MtaA activity was more rapid in the presence of NaHS/H2S than in the presence of EDTA. Even at low NaHS/H2S concentration a constant activity was reached already after several minutes of incubation. The 1 mL incubation mixtures contained: 50 mm Mops/KOH pH 7.0, 50 mm methylcob(III)alamin, 8.8 mg holoprotein and EDTA, nitrilotriacetic acid, NaHS/H2S and/or ZnCl2 at the concentrations indicated. At the times indicated the temperature was increased to 37 8C, coenzyme M (0.5 mm) was added and the MtaA activity determined by following the decrease in methylcob(III)alamin concentration photometrically at 520 nm.
MtaBC complex under the same conditions (unpublished results). Binding of Zn21 to MtaA In order to estimate how tight zinc is bound to MtaA, the active enzyme was incubated at pH 7.0 in the presence of zinc complexing agents such as EDTA (Kd 10216.7 m) or nitrilotriacetic acid (Kd 10210.7 m) [19] and the rate and degree of inactivation were determined (Fig. 4A). In the presence of 1 mm EDTA < 75% of the activity was lost while 1 mm nitrilotriacetic acid had almost no effect on the MtaA activity. In the presence of 1 mm EDTA and 0.1 mm ZnCl2 only < 50% of the activity was lost. Inactivation was at least partially reversible as indicated by the finding that after 75% inactivation of the enzyme in the presence of 1 mm EDTA the activity increased again upon addition of 0.1 mm ZnCl2 (Fig. 4A). Therefore, from the activity data and the concentration of noncomplexed zinc in the assay an apparent dissociation constant Kd of the MtaA±zinc complex can be estimated taking into account that the activity approximately reflects the zinc content of MtaA. The concentration of free Zn21 in the assays was calculated from the total EDTA concentration and the total Zn21 concentration [20] considering that at pH 7.0 EDTA is mainly present in its monoprotonated form (pK1 2.0; pK2 2.7; pK3 6.2; pK4 10.3) [19] and
that Zn21 is most tightly complexed by the unprotonated form (Kd 10216.7 M). At pH 7.0, a total EDTA concentration of 1 mm and a total zinc concentration of 0.1 mm the concentration of free Zn21 is < 10214 m. At this concentration MtaA was 50% active indicating that the apparent dissociation constant of the MtaA±zinc complex is < 10214 m. We tried to obtain an independent estimate of the Kd by measuring the activity of MtaA after incubation in the presence of NaHS/H2S (pK1 6.99 and pK2 12.9). The solubility product of ZnS (Zn21 1 S2± N ZnS) is 4.5 10224 m [21] and thus much lower than the dissociation constant Ks 10216.7 m of the zinc±EDTA complex. In Fig. 4B the effect of NaHS/H2S on the MtaA activity is compared with that of EDTA. The enzyme was incubated at pH 7.0 and 20 8C for 1 h in the presence of NaHS/H2S or EDTA at the concentrations indicated and the MtaA activity was then determined. Fifty percent loss of activity was observed at a NaHS/H2S concentration of < 4 mm. At this concentration the free Zn21 concentration is < 10215 m. At NaHS/H2S concentrations above 4 mm the activity decreased somewhat less than the predicted free-zinc concentration, which may be because at very low free Zn21 concentrations the rate of ZnS formation becomes very low. An alternative explanation, that NaHS/H2S at high concentrations was used as substrate, was ruled out by showing that demethylation of methylcobalamin was dependent on the presence of coenzyme M.
q FEBS 2000
Involvement of zinc in coenzyme M methylation (Eur. J. Biochem. 267) 2503
DISCUSSION The results clearly indicate that methylcobalamin:coenzyme M methyltransferase (MtaA) requires zinc or cobalt for activity and that these transition metals are involved in coenzyme M activation by lowering the microscopic pKa of its thiol group from . 9 to , 7 as determined by proton release at pH 7.7 following coenzyme M binding. The catalytic role of zinc in MtaA is thus identical to that shown previously for zinc in cobalamin-dependent methionine synthase (MetH) [13,14,22], cobalamin-independent methionine synthase (MetE) [22±24] and protein farnesyl transferase [25±30], and proposed for zinc in the E. coli Ada protein [31,32], betaine:homocysteine S-methyltransferase (BHMT) [33,34], S-methylmethionine:homocysteine methyltransferase (YagD) [35,36] and epoxyalkane:coenzyme M transferase [37±39]. What these zinc enzymes all have in common is that they catalyze the alkylation of a thiol group [26,40]. The catalytic role of zinc in MtaA is also in agreement with model studies of the alkylation of zinc thiolate complexes [41±44]. Evidence is available that MetH, BHMT and YagD have the zinc-binding motif C-Xn-GGCC, MetE, epoxyalkane: coenzyme M transferase and MtaA the zinc binding motif H-X-C-Xn-C, the Ada protein the zinc binding motif C-X3-CX26-C-X2-C and the protein farnesyl transferase the zinc binding motif D-X-C-X49262-H [23,26,35,40]. The dissociation constant Kd of the enzyme±zinc complex has recently been estimated to be , 10216 m for MetE from the finding that the activity of MetE was not affected by mm concentrations of EDTA (Kd 10217.6 m) [23,24]. For MtaA we found an apparent Kd of < 10214 m determined with EDTA and of < 10215 m determined with S2± . The apparent Kd, which was calculated from the specific activity before and after incubation with EDTA or NaHS/H2S and from the free zinc concentration is, however, very probably larger than the true Kd since any inhibition of enzyme activity by the complexing agents not due to reversible dissociation of the enzyme±zinc complex leads to an apparent increase in the Kd. This has to be considered particularly in the case of EDTA. It is known that EDTA tends to bind nonspecifically to proteins [45] and by that to inhibit enzyme activity. This may explain why MtaA activity was less affected by NaHS than by EDTA despite the fact that S2± has the higher affinity for Zn21. M. barkeri can grow at pH 7.0 on methanol media containing concentrations of NaHS/H2S up to 7 mm, which is probably also the concentration within the cells. At 7 mm the concentration of free Zn21 is < 10215 m. Zinc enzymes in M. barkeri should, therefore, have Kd values of , 10215 m in order to be active during growth in the presence of NaHS/H2S. In this respect it is of interest that the growth rate of M. barkeri does decrease with increasing NaHS/H2S concentrations from 0.17 h21 at 1.4 mm, 0.08 h21 at 4.2 mm to 0.02 h21 at 7 mm (K. Suaer & R. K. Thauer, unpublished results). The growth inhibition indicates that M. barkeri does not use cobalt instead of zinc when growing in the presence of NaHS/H2S despite the fact that under these conditions the free Co21 concentration is 20 times higher than that of Zn21 due to the much higher solubility product of CoS (8.0 10223 m) [46]. In a previous publication we reported the dissociation constant of the MtaA±zinc complex to be 1029.6 m [4]. This value was obtained from activity measurements after incubation of the enzyme in the presence of 20 mm nitrilotriacetic acid and different concentrations of ZnCl2. Fifty percent activity was observed at a concentration of free Zn21 of 1029.6 m and of free
Co21 of 1029.3 m [4]. After incubation the maximally recovered activity was, however, only , 0.1% of the activity of MtaA before inactivation with nitrilotriacetic acid indicating that under the experimental conditions employed most of the enzyme was irreversibly inactivated. This was not considered when interpreting these results.
ACKNOWLEDGEMENTS This work was supported by the Max-Planck-Gesellschaft, by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. We want to thank Prof. Dr R. J. P. Williams (Oxford) for drawing to our attention that the intracellular concentration of free zinc in strict anaerobes, which thrive in the presence of H2S, should be in the order of 10215 m.
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