Vol. 172, No. 8
JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 44564463
0021-9193/90/084456-08$02.00/0 Copyright ) 199, American Society for Microbiology
Formate Dehydrogenase from the Methane Oxidizer Methylosinus trichosporium OB3b DUANE C. YOCH,* YUNG-PIN CHEN, AND MICHAEL G. HARDIN Department ofBiological Sciences, University of South Carolina, Columbia, South Carolina 29208 Received 19 December 1989/Accepted 4 May 1990
Formate dehydrogenase (NAD+ depet) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isobted anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 celluloe and Sephacryl S-300 colns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the a-subunit was 53,800 ± 2,800, and that of the a-subunit was 102,600 ± 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent a2j2 arrangemnt. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were d d. No other metals were found in stoichimetr amounts. When the enzyme was isolated aerobicaly, there was no cofactor requiremet for NAD reduction; however, when ioated annaebically, activity was 80 to 90% dependent on the addition of favin mononucleotide (FMN) to the rmction Ixture. Furthermore, the addition of formate to an active, anoxc soution of formate dehydrogenase rapidly inactivated it in the absence of an e on por; this activity couid be reconstituted approximatly 85% by 50 nM FMN. Flavin adenine dinucltide could not replace FMN in reconstituting enzyme activity. The K,,s of formate dehydrogense for formate, NAD, and FMN were 146, 200, and 0.02 FM, respectivdy. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inacvated under anoxic conditions by formate and reactivated by FMN. The evolutionary slnficance of this sindilarity is discussed.
Among the large number of microorganisms that have formate dehydrogenase, the NAD-linked enzyme (EC 1.2.1.2) has been isolated from both facultative methylotrophic bacteria (3, 13) and several yeast species (20; also reference 4 and references therein). There is a great deal of heterogeneity in the structure and composition of bacterial formate dehydrogenases. They range from molecules with two identical subunits and no prosthetic group, found in facultative methylotrophic strains of Moraxella (30) and in Pseudomonas sp. strain 101 (formerly Achromobacter) (13, 24), to those isolated from the obligate anaerobes Clostridium thermoaceticum (33) and Methanococcus vannelli (20), which have three different metals in their prosthetic group(s). Escherichia coli formate dehydrogenase contains three different subunits, metals, and a heme group (14). In methylotrophs, formate dehydrogenase is a critical component involved in the oxidative metabolism of methane and methanol. The NADH generated by this enzyme is, in some cases, believed to be the cell's only source of reductant (2). In Methylosinus trichosporium, NAD-linked formate dehydrogenase functions as an in vitro electron donor to methane monooxygenase (32) and indirectly to nitrogenase via a ferredoxin-NAD+ reductase and ferredoxin (9, 10) (Fig. 1). Although formate dehydrogenase plays an important role in the metabolism of nonfacultative methylotrophs (and obligate methanotrophs), this enzyme has not yet been isolated from either of these groups of bacteria. In this study we purified the formate dehydrogenase from the obligate methanotroph M. trichosporium and report on its structural and regulatory characteristics.
*
MATERIALS AND METHODS Growth conditions. M. trichosporium OB3b cells used for formate dehydrogenase preparations were batch cultured in 9-liter carboys on nitrate-containing medium (11) as described previously (10). In these cultures, the air-methane mixture (5:1) was dispersed by passing it through stone diffusers and stirring the culture slowly with a magnetic bar. Our cultures did not attain a high density (80 to 120 Klett units, no. 66 filter) in these batch cultures and were harvested by centrifugation after 6 days of growth. After harvesting, the cells were immediately frozen (unwashed) in liquid nitrogen until needed. Formate dehydrogenase assays. Unless otherwise indicated, formate dehydrogenase activity was measured at room temperature (22 to 25°C) by monitoring the reduction of NAD (e340, 6.2 mM-' cm-') with a Perkin-Elmer Lambda 3 spectrophometer under aerobic conditions. The reaction mixture (in 1 ml) contained 50 mM sodium phosphate buffer (pH 7), 20 mM sodium formate, 0.5 mM NAD, and 2.5 ,uM flavin mononucleotide (FMN) unless indicated. The reaction was started by the addition of enzyme to the cuvette. One unit of formate dehydrogenase activity was defined as the amount of enzyme that reduced 1 ,umol of NAD per min. Specific activities are reported as units per milligram of
protein.
Formate dehydrogenase activity was also assayed by monitoring the reduction of 2,6-dichlorophenol indophenol (DCIP). The assay mixture (in a final volume of 2 ml) contained enzyme, 25 mM Tris buffer (pH 7.4), 25 ,uM DCIP, 2.5 p,M phenazine methosulfate, and 20 mM formate. The assays were carried out anaerobically in stoppered cuvettes under an atmosphere of argon by monitoring the reduction of DCIP (e6w, 19.1 mM-1 cm-'). Purification of formate dehydrogenase. The enzyme was purified anaerobically from 75-g samples of cells that had
Corresponding author. 4456
VOL. 172, 1990 C
MMO
LIDH2
CH3
NAD
FORMATE DEHYDROGENASE FROM M. TRICHOSPORIUM MDH POO
FADH
HCHO ND
POOH2
NAD
NHCOOH NA
2
FDH_c 0
NAD
genase and its reconstitution
2
NADH2
*FNR Fd
;(N2
2NH3 FIG. 1. Oxidation of methane in M. trichosporium OB3b coupled to the reduction of N2. Enzyme abbreviations: MMO, methane monooxygenase; MDH, methanol dehydrogenase; FADH, formaldehyde dehydrogenase; FDH, formate dehydrogenase; FNR, ferredoxin-NAD+ reductase; Fd, ferredoxin; N2ase, nitrogenase. PQQ and PQQH2 are the oxidized and reduced forms, respectively, of pyrroloquinoline quinone.
been stored in liquid N2. The buffer used in all of the purification steps was composed of 50 mM sodium phosphate adjusted to pH 7.2, 10% (vol/vol) glycerol, and the proteinase inhibitors benzamidine (1 mM) and phenylmethylsulfonyl fluoride (0.1 mM). This mixture is referred to as the buffer in descriptions of the purification of formate dehydrogenase. When the enzyme was prepared anaerobically, the buffer was degassed by repeated cycles of evacuation and refilling of a side-arm flask with argon. Just before use, glucose (1 g/liter) and glucose oxidase (8 mg/liter) were added to the buffer as an oxygen scavenger system. Extracts were prepared by anaerobic sonication as described previously (8). Following centrifugation for 20 min at 27,000 rpm, 1 mg of DNase I was added to the supernatant extract. Formate dehydrogenase was collected by passing the crude extract over a bed (4.5 by 6 cm) of DE-52-cellulose in a 60-cm chromatography column constantly flushed with argon. All the extract was applied to the column at once and maintained under this argon cover. The column was washed with degassed buffer; formate dehydrogenase was eluted as a sharp brown band, followed by a smaller band of activity with buffer containing 0.15 M NaCl. DE-52 chromatography was performed on the major peak of activity by diluting it threefold with buffer and applying it to another column (1.7 by 27 cm). The enzyme was eluted with 200 ml of a 0 to 0.3 M NaCl gradient. Because the enzyme sometimes lost its FMN cofactor at this stage, 1 j±M FMN was routinely added to the reaction mixture before these fractions were assayed. DE-52-cellulose fractions with the highest activity were diluted 1:1 with buffer and applied to a DEAE-Sephadex A50 column (0.9 by 28 cm) that had previously been equilibrated with degassed buffer. The column was eluted with 180 ml of a gradient of NaCl from 0.1 to 0.45 M in buffer. A major peak of activity (60% of the total) usually preceded a minor band at this stage of purification. The minor band (the lower-Mr formate dehydrogenase species) was not purified beyond this point. The major peak of activity was concentrated on a Diaflow (Millipore Corp.) apparatus with a PM30 membrane. The concentrated enzyme was applied to a Sephacryl S-200 column (0.9 by 53 cm) eluted with anaerobic buffer containing 200 mM NaCl. Most (90%) of the activity was in a high-Mr form. These fractions were finally applied to a second DEAE-Sephadex A-50 column which was equilibrated and eluted the same as the first. A number of fractions from this column had only minor impurities and were used for subunit analysis. Formate-induced enzyme inactivation of formate dehydro-
4457
by FMN. Formate
dehydrogewhich was prepared aerobically yielded the active form of the enzyme. It was inactivated in a 50-,u reaction mixture in a stoppered degassed microfuge tube containing 10 of glucose (1 g/liter) and glucose oxidase 8 mg and 0.5 RImM sodium formate. After 1.5 min, 0.95 ml of the reaction mixture for NAD reduction (with or without FMN) was added to the microfuge tube, and the solution was transferred to a cuvette to monitor NAD reduction. Since the flavin cofactor released from formate dehydrogenase was diluted out in this reaction mixture, it did not have to be removed from the formate-pretreated enzyme in order to demonstrate that the enzyme had been inactivated. Polyacrylamide gel electrophoresis. Nondissociating discontinuous polyacrylamide tube gel electrophoresis was performed by the method of Hames (18). In this system, the gel buffer was pH 8.8 and the reservoir buffer was pH 8.3. Gels were stained for protein with 0.1% Coomassie blue R-250 in 25% methanol and 10% acetic acid, or for formate dehydrogenase activity by being placed in a solution of formate and triphenyltetrazolium chloride, as described by Yamamoto et al. (33). When activity stains were desired, the upper reservoir buffer (which was enclosed) was first degassed and then bubbled continuously with argon, while the gels were preelectrophoresed for 1 h. To rapidly remove the gels from the tubes, they were frozen in liquid N2 for a minute to shatter the glass and then placed in test tubes containing degassed activity-staining solution. These tubes were stoppered, evacuated, and refilled with argon. To determine the Mr of the formate dehydrogenase subunits, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed with 10% polyacrylamide slab gels and a Laemmli-type discontinuous-system buffer run at pH 8.6 (18). Other analytical methods. Protein was determined by the Folin-phenol method (26) with bovine serum albumin as the standard. The method of Andrews (1) was used to estimate the Mr of formate dehydrogenase by the gel filtration method on Sephacryl S-300 which was equilibrated and in 0.1 M phosphate buffer, pH 7.2, containing 0.1developed M KCI. Trace metals were determined by inductively coupled plasma emission spectrascopy on a Jarrell-Ash model 965 ICP at the Institute of Ecology, University of Georgia, Athens. The sensitivity for iron was 0.05 ppm. Nonheme (ferrous) iron was also determined by the bathophenanthroline method (18). Anabaena ferredoxin (2 g-atoms of Fe per mol) was used as a standard. Acid-labile sulfide levels were determined by the method of Fogo and Popowsky (15) as modified by Brumby et al. (7). nase
RESULTS Different Mr forms of formate dehydrogenase. When crude extracts were electrophoresed on native disk gels and examined by an activity stain technique, the major band in front of a minor, slower-migrating band of higher migrated Mr (data not shown). Furthermore, activity profiles from chromatography columns often, but not always, showed two peaks of NAD+-reducing activity. For example, on a DE-52-cellulose column, the major activity band eluted with a low concentration of NaCl and a minor peak of activity eluted with a higher salt concentration (Fig. 2). When the main peak from the DE-52-cellulose column was chromatographed on a Sephacryl S-300 column, the major formate dehydrogenase component was the high-Mr component. We do not know at this time what controls the equilibrium between the two Mr
4458
J. BACTERIOL.
YOCH ET AL.
4
=
20-
tj.15M NaCI
O.1M NaCI
o10
LL
0
20
10
Fraction number FIG. 2. DE-52-cellulose chromatography showing two molecular forms of formate dehydrogenase (FDH) in crude extract. This column (1.5 by 12 cm) had previously been washed with 200 ml each of 20 mM phosphate buffer containing 0 and 0.05 M NaCl. Formate dehydrogenase was eluted with buffer containing 0.1 M NaCl as indicated. All fractions were assayed in the presence of FMN.
forms of formate dehydrogenase, nor are we prepared to say which form predominates in vivo. Purification of formate dehydrogenase. The purification of formate dehydrogenase from M. trichosporium, which resulted in a 130-fold increase in NAD+-reducing activity, is summarized in Table 1. The methods were anaerobic but did not include the use of a glove box. Column fractions were collected in air, evacuated and refilled with argon several times, and stored on ice. Purification under anaerobic conditions was always accompanied by loss of activity, but this activity could be stimulated 4- to 10-fold by the addition of FMN to the reaction mixture. This (reversible) loss of activity was greatest when an oxygen scavenger such as glucose or glucose oxidase was added to the buffers. When the enzyme was purified aerobically, there was a much smaller loss of activity, but it could not be recovered by added FMN. The flavin requirement for this enzyme is discussed in detail below. As shown in Table 1, a large loss in activity occurred on the first DE-52-cellulose column. This loss occurred in both aerobic and anoxic preparations. After the work here had been completed, we found that adding Fe2+ [100 ,uM Fe(NH4)2SO4] and a thiol reducing agent (1 mM cysteine) to the buffer increased the recovery of formate dehydrogenase activity at this step from 20% to approximately 70%. These reagents appear to reconstitute the Fe/S center(s) of formate dehydrogenase, just as they did for the hydrolyase portion of the methane monooxygenase of this organism (16). The specific activity of formate dehydrogenase in crude
FIG. 3. Polyacrylamide gel electrophoresis of purified formate dehydrogenase. (A) Lanes 1 and 2, Native disk gels stained for formate dehydrogenase activity and for protein with Coomassie blue R-250, respectively. (B) Lane 3, SDS-polyacrylamide gel stained for protein with Coomassie R-250 (5 ,ug of protein added). Lane 4, Molecular mass markers: myosin (205 kDa), E. coli ,B-galactosidase (116 kDa), rabbit muscle phosphorylase B (97 kDa), bovine serum albumin (66 kDa), hen egg albumin (45 kDa), and carbonic anhydrase (29 kDa). extracts ranged from 0.12 to 0.3 ,umol of NAD+ reduced per
min per mg of protein. The purification process (Table 1) yielded near homogeneous enzyme having a specific activity of approximately 32 U/mg. Figure 3A, lane 1, is an activity stain of formate dehydrogenase on a native polyacrylamide gel. Lane 2 shows a Coomassie stain of the enzyme following purification; it appears to be greater than 90% pure. Molecular mass and subunit structure. The molecular mass of M. trichosporium formate dehydrogenase determined on a calibrated Sephacryl S-300 column (0.9 by 58 cm) was approximately 315 kilodaltons (kDa) (Fig. 4). This value is an average from values for relatively pure samples applied to the column and for a preparation that had only one previous step of purification. The latter sample was used to determine whether the two size species of formate dehydrogenase that often exist early in the purification procedure were comparable in size to the purified components. They seemed to be, as sizes of 309 and 155 kDa were measured. The 315-kDa form of formate dehydrogenase was demonstrated by electrophoresis in a 10% polyacrylamide gel in the presence of 0. 1% SDS to have two subunits (Fig. 3B, lane 3). The a-subunit had a mean molecular weight of 102,600 3,900, and the P-subunit was 54,800 + 2,800 (five determinations). Since the low-Mr form of formate dehydrogenase also showed the same subunit size pattern (data not shown), this suggests that the enzyme is an a,B heterodimer and that the high-Mr form is an (X2P2 tetramer. The physical and
TABLE 1. Purification of M. trichosporium formate dehydrogenase Purification step
Crude extract First DE-52-cellulose Second DE-52 cellulose DEAE-Sephadex A-50 First Sephacryl S-200 Second DEAE-Sephacryl A50
Activity
Vol
Protein Concn
Total
Concn
Total
Yield
Sp act
Purification
(ml)
(mg/ml)
(mg)
(U/ml)
(U)
(%)
(U/mg)
(fold)
100 18 23 12 5 1
0.25 2.2 9.7 17.7 26.6 32.3
1 8.6 39 71 106 130
490 87 14 8 5.9 2.0
2.0 1.1 2.0 1.0 0.35 0.09
980 96 28 8 2 1.8
2.3 2.3 19.4 17.7 9.2 5.8
1,153 207 272 142 54 11.6
VOL. 172, 1990
FORMATE DEHYDROGENASE FROM M. TRICHOSPORIUM
800
z 0. z -r
tz -'I
0
600 coO
z
(0
4001-
0.
0.
(4)
P:
kinetic characteristics of the enzyme are summarized in Table 2, where they are compared with formate dehydrogenases from "Pseudomonas oxalaticus" and a Moraxella species. The metal cofactor. M. trichosporium formate dehydrogenase is a brown protein which has a peak at 278 nm and a rather nondescript shoulder in the 300- to 400-nm region when isolated in the deflavo form. The addition of 5 mM sodium formate bleached the enzyme to about one-fourth the extent of dithionite (data not shown). The amounts of iron, nickel, and molybdenum have been determined on several preparations of formate dehydrogenase. It has not been practical to analyze preparations of very high activity (>30 U/mg) because of the relatively small amount of enzyme that was available. Based on their purity and metal content, we calculated the range of metal concentrations in several preparations (Table 2). Nonheme iron was the major metallic component, with values ranging between 11 and 18 g-atom per mol of enzyme (Mr 315,000). Inorganic sulfur was also present, with values between 15 and 20 mol/mol of enzyme being obtained. There were only traces of molybdenium and nickel. Selenium, which is present in this enzyme from a number of anaerobes, was not analyzed. Reversible inactivation and reconstitution of activity with FMN. When formate dehydrogenase from M. tnchosporium
0',
MD
w
0-t
t:
cz) z
z
z
C
Z
0
z
I
t::
-I
0j
00~ o0
3 -FM
LI.
Fraction number FIG. 5. Effect of added FMN on formate dehydrogenase (FDH) activity following chromatography on DE-52-cellulose. The column (1.5 by 70 cm) (which followed the DE-52-cellulose collecting column) was eluted with 200 ml of buffer containing a 0 to 0.3 M NaCl gradient. The inset shows the kinetics of the enzyme in the peak fraction when 0.5 ,uM FMN was added at the times indicated by the arrows. Protein concentration was 50 ,ug/ml.
was prepared under aerobic conditions, the enzyme was fully active as isolated. However, when this enzyme was purified anaerobically, we found that these preparations lost most of their activity following an early chromatography
z
30-
40
40 o;
~20 -20
1
U.
I~~~~~~~~~~~~~~~~~~~~ 0 1
2 34
Minutes
5 0
20
Formate
40
(piM)
l1
20 30405
FMN (nM)
FIG. 6. Inactivation and FMN-mediated reconstitution of formate dehydrogenase (FDH) activity. Formate-induced inactivation was carried out for 2 min (or in panel A for the time indicated) in a 50-pd reaction mixture in a microfuge tube containing enzyme (as indicated), (see formate, and a glucose-glucose oxidase 02 scavenger. After this preincubation, the normal formate dehydrogenase assay mixture Materials and Methods), with or without a flavin cofactor (as indicated), was added to this solution, which was then transferred to a cuvette to monitor NAD reduction. (A) Time course of inactivation by formate added at time zero (0), and the effect of 2 ,uM FMN (0) or 2 p.M FAD (A) in reconstituting this activity. Formate dehydrogenase, 19 ,ug of protein. (B) Effect of sodium formate concentration on the reversible inactivation of formate dehydrogenase. The enzyme (19 p.g of protein) was inactivated as described above with the concentration of formate indicated. After 2 min, the 50-pul preincubation mixture was added to 1 ml of assay mixture to determine the extent of inactivation. (C) Effect of FMN concentration on reactivating formate-inactivated formate dehydrogenase. A tube of formate dehydrogenase of high specific activity was inactivated for 2 min with 0.5 mM formate as described above. Samples of enzyme (1.3 jig of protein) were removed and added to the assay mixture containing FMN at the concentrations indicated. The dashed line represents the initial activity of the formate dehydrogenase, or the point at which the enzyme would have recovered 100% of its original activity.
FORMATE DEHYDROGENASE FROM M. TRICHOSPORIUM
VOL. 172, 1990
(a)
T
AA 340
g -
0.1
(c) )
1 min
(b)
FIG. 7. Formate-induced inactivation of "P. oxalaticus" formate dehydrogenase and its reactivation by FMN. "P. oxalaticus"
formate dehydrogenase was obtained from Sigma Chemical Co. (St. Louis, Mo.) and used without further purification (specific activity,
JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 44564463
0021-9193/90/084456-08$02.00/0 Copyright ) 199, American Society for Microbiology
Formate Dehydrogenase from the Methane Oxidizer Methylosinus trichosporium OB3b DUANE C. YOCH,* YUNG-PIN CHEN, AND MICHAEL G. HARDIN Department ofBiological Sciences, University of South Carolina, Columbia, South Carolina 29208 Received 19 December 1989/Accepted 4 May 1990
Formate dehydrogenase (NAD+ depet) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isobted anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 celluloe and Sephacryl S-300 colns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the a-subunit was 53,800 ± 2,800, and that of the a-subunit was 102,600 ± 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent a2j2 arrangemnt. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were d d. No other metals were found in stoichimetr amounts. When the enzyme was isolated aerobicaly, there was no cofactor requiremet for NAD reduction; however, when ioated annaebically, activity was 80 to 90% dependent on the addition of favin mononucleotide (FMN) to the rmction Ixture. Furthermore, the addition of formate to an active, anoxc soution of formate dehydrogenase rapidly inactivated it in the absence of an e on por; this activity couid be reconstituted approximatly 85% by 50 nM FMN. Flavin adenine dinucltide could not replace FMN in reconstituting enzyme activity. The K,,s of formate dehydrogense for formate, NAD, and FMN were 146, 200, and 0.02 FM, respectivdy. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inacvated under anoxic conditions by formate and reactivated by FMN. The evolutionary slnficance of this sindilarity is discussed.
Among the large number of microorganisms that have formate dehydrogenase, the NAD-linked enzyme (EC 1.2.1.2) has been isolated from both facultative methylotrophic bacteria (3, 13) and several yeast species (20; also reference 4 and references therein). There is a great deal of heterogeneity in the structure and composition of bacterial formate dehydrogenases. They range from molecules with two identical subunits and no prosthetic group, found in facultative methylotrophic strains of Moraxella (30) and in Pseudomonas sp. strain 101 (formerly Achromobacter) (13, 24), to those isolated from the obligate anaerobes Clostridium thermoaceticum (33) and Methanococcus vannelli (20), which have three different metals in their prosthetic group(s). Escherichia coli formate dehydrogenase contains three different subunits, metals, and a heme group (14). In methylotrophs, formate dehydrogenase is a critical component involved in the oxidative metabolism of methane and methanol. The NADH generated by this enzyme is, in some cases, believed to be the cell's only source of reductant (2). In Methylosinus trichosporium, NAD-linked formate dehydrogenase functions as an in vitro electron donor to methane monooxygenase (32) and indirectly to nitrogenase via a ferredoxin-NAD+ reductase and ferredoxin (9, 10) (Fig. 1). Although formate dehydrogenase plays an important role in the metabolism of nonfacultative methylotrophs (and obligate methanotrophs), this enzyme has not yet been isolated from either of these groups of bacteria. In this study we purified the formate dehydrogenase from the obligate methanotroph M. trichosporium and report on its structural and regulatory characteristics.
*
MATERIALS AND METHODS Growth conditions. M. trichosporium OB3b cells used for formate dehydrogenase preparations were batch cultured in 9-liter carboys on nitrate-containing medium (11) as described previously (10). In these cultures, the air-methane mixture (5:1) was dispersed by passing it through stone diffusers and stirring the culture slowly with a magnetic bar. Our cultures did not attain a high density (80 to 120 Klett units, no. 66 filter) in these batch cultures and were harvested by centrifugation after 6 days of growth. After harvesting, the cells were immediately frozen (unwashed) in liquid nitrogen until needed. Formate dehydrogenase assays. Unless otherwise indicated, formate dehydrogenase activity was measured at room temperature (22 to 25°C) by monitoring the reduction of NAD (e340, 6.2 mM-' cm-') with a Perkin-Elmer Lambda 3 spectrophometer under aerobic conditions. The reaction mixture (in 1 ml) contained 50 mM sodium phosphate buffer (pH 7), 20 mM sodium formate, 0.5 mM NAD, and 2.5 ,uM flavin mononucleotide (FMN) unless indicated. The reaction was started by the addition of enzyme to the cuvette. One unit of formate dehydrogenase activity was defined as the amount of enzyme that reduced 1 ,umol of NAD per min. Specific activities are reported as units per milligram of
protein.
Formate dehydrogenase activity was also assayed by monitoring the reduction of 2,6-dichlorophenol indophenol (DCIP). The assay mixture (in a final volume of 2 ml) contained enzyme, 25 mM Tris buffer (pH 7.4), 25 ,uM DCIP, 2.5 p,M phenazine methosulfate, and 20 mM formate. The assays were carried out anaerobically in stoppered cuvettes under an atmosphere of argon by monitoring the reduction of DCIP (e6w, 19.1 mM-1 cm-'). Purification of formate dehydrogenase. The enzyme was purified anaerobically from 75-g samples of cells that had
Corresponding author. 4456
VOL. 172, 1990 C
MMO
LIDH2
CH3
NAD
FORMATE DEHYDROGENASE FROM M. TRICHOSPORIUM MDH POO
FADH
HCHO ND
POOH2
NAD
NHCOOH NA
2
FDH_c 0
NAD
genase and its reconstitution
2
NADH2
*FNR Fd
;(N2
2NH3 FIG. 1. Oxidation of methane in M. trichosporium OB3b coupled to the reduction of N2. Enzyme abbreviations: MMO, methane monooxygenase; MDH, methanol dehydrogenase; FADH, formaldehyde dehydrogenase; FDH, formate dehydrogenase; FNR, ferredoxin-NAD+ reductase; Fd, ferredoxin; N2ase, nitrogenase. PQQ and PQQH2 are the oxidized and reduced forms, respectively, of pyrroloquinoline quinone.
been stored in liquid N2. The buffer used in all of the purification steps was composed of 50 mM sodium phosphate adjusted to pH 7.2, 10% (vol/vol) glycerol, and the proteinase inhibitors benzamidine (1 mM) and phenylmethylsulfonyl fluoride (0.1 mM). This mixture is referred to as the buffer in descriptions of the purification of formate dehydrogenase. When the enzyme was prepared anaerobically, the buffer was degassed by repeated cycles of evacuation and refilling of a side-arm flask with argon. Just before use, glucose (1 g/liter) and glucose oxidase (8 mg/liter) were added to the buffer as an oxygen scavenger system. Extracts were prepared by anaerobic sonication as described previously (8). Following centrifugation for 20 min at 27,000 rpm, 1 mg of DNase I was added to the supernatant extract. Formate dehydrogenase was collected by passing the crude extract over a bed (4.5 by 6 cm) of DE-52-cellulose in a 60-cm chromatography column constantly flushed with argon. All the extract was applied to the column at once and maintained under this argon cover. The column was washed with degassed buffer; formate dehydrogenase was eluted as a sharp brown band, followed by a smaller band of activity with buffer containing 0.15 M NaCl. DE-52 chromatography was performed on the major peak of activity by diluting it threefold with buffer and applying it to another column (1.7 by 27 cm). The enzyme was eluted with 200 ml of a 0 to 0.3 M NaCl gradient. Because the enzyme sometimes lost its FMN cofactor at this stage, 1 j±M FMN was routinely added to the reaction mixture before these fractions were assayed. DE-52-cellulose fractions with the highest activity were diluted 1:1 with buffer and applied to a DEAE-Sephadex A50 column (0.9 by 28 cm) that had previously been equilibrated with degassed buffer. The column was eluted with 180 ml of a gradient of NaCl from 0.1 to 0.45 M in buffer. A major peak of activity (60% of the total) usually preceded a minor band at this stage of purification. The minor band (the lower-Mr formate dehydrogenase species) was not purified beyond this point. The major peak of activity was concentrated on a Diaflow (Millipore Corp.) apparatus with a PM30 membrane. The concentrated enzyme was applied to a Sephacryl S-200 column (0.9 by 53 cm) eluted with anaerobic buffer containing 200 mM NaCl. Most (90%) of the activity was in a high-Mr form. These fractions were finally applied to a second DEAE-Sephadex A-50 column which was equilibrated and eluted the same as the first. A number of fractions from this column had only minor impurities and were used for subunit analysis. Formate-induced enzyme inactivation of formate dehydro-
4457
by FMN. Formate
dehydrogewhich was prepared aerobically yielded the active form of the enzyme. It was inactivated in a 50-,u reaction mixture in a stoppered degassed microfuge tube containing 10 of glucose (1 g/liter) and glucose oxidase 8 mg and 0.5 RImM sodium formate. After 1.5 min, 0.95 ml of the reaction mixture for NAD reduction (with or without FMN) was added to the microfuge tube, and the solution was transferred to a cuvette to monitor NAD reduction. Since the flavin cofactor released from formate dehydrogenase was diluted out in this reaction mixture, it did not have to be removed from the formate-pretreated enzyme in order to demonstrate that the enzyme had been inactivated. Polyacrylamide gel electrophoresis. Nondissociating discontinuous polyacrylamide tube gel electrophoresis was performed by the method of Hames (18). In this system, the gel buffer was pH 8.8 and the reservoir buffer was pH 8.3. Gels were stained for protein with 0.1% Coomassie blue R-250 in 25% methanol and 10% acetic acid, or for formate dehydrogenase activity by being placed in a solution of formate and triphenyltetrazolium chloride, as described by Yamamoto et al. (33). When activity stains were desired, the upper reservoir buffer (which was enclosed) was first degassed and then bubbled continuously with argon, while the gels were preelectrophoresed for 1 h. To rapidly remove the gels from the tubes, they were frozen in liquid N2 for a minute to shatter the glass and then placed in test tubes containing degassed activity-staining solution. These tubes were stoppered, evacuated, and refilled with argon. To determine the Mr of the formate dehydrogenase subunits, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed with 10% polyacrylamide slab gels and a Laemmli-type discontinuous-system buffer run at pH 8.6 (18). Other analytical methods. Protein was determined by the Folin-phenol method (26) with bovine serum albumin as the standard. The method of Andrews (1) was used to estimate the Mr of formate dehydrogenase by the gel filtration method on Sephacryl S-300 which was equilibrated and in 0.1 M phosphate buffer, pH 7.2, containing 0.1developed M KCI. Trace metals were determined by inductively coupled plasma emission spectrascopy on a Jarrell-Ash model 965 ICP at the Institute of Ecology, University of Georgia, Athens. The sensitivity for iron was 0.05 ppm. Nonheme (ferrous) iron was also determined by the bathophenanthroline method (18). Anabaena ferredoxin (2 g-atoms of Fe per mol) was used as a standard. Acid-labile sulfide levels were determined by the method of Fogo and Popowsky (15) as modified by Brumby et al. (7). nase
RESULTS Different Mr forms of formate dehydrogenase. When crude extracts were electrophoresed on native disk gels and examined by an activity stain technique, the major band in front of a minor, slower-migrating band of higher migrated Mr (data not shown). Furthermore, activity profiles from chromatography columns often, but not always, showed two peaks of NAD+-reducing activity. For example, on a DE-52-cellulose column, the major activity band eluted with a low concentration of NaCl and a minor peak of activity eluted with a higher salt concentration (Fig. 2). When the main peak from the DE-52-cellulose column was chromatographed on a Sephacryl S-300 column, the major formate dehydrogenase component was the high-Mr component. We do not know at this time what controls the equilibrium between the two Mr
4458
J. BACTERIOL.
YOCH ET AL.
4
=
20-
tj.15M NaCI
O.1M NaCI
o10
LL
0
20
10
Fraction number FIG. 2. DE-52-cellulose chromatography showing two molecular forms of formate dehydrogenase (FDH) in crude extract. This column (1.5 by 12 cm) had previously been washed with 200 ml each of 20 mM phosphate buffer containing 0 and 0.05 M NaCl. Formate dehydrogenase was eluted with buffer containing 0.1 M NaCl as indicated. All fractions were assayed in the presence of FMN.
forms of formate dehydrogenase, nor are we prepared to say which form predominates in vivo. Purification of formate dehydrogenase. The purification of formate dehydrogenase from M. trichosporium, which resulted in a 130-fold increase in NAD+-reducing activity, is summarized in Table 1. The methods were anaerobic but did not include the use of a glove box. Column fractions were collected in air, evacuated and refilled with argon several times, and stored on ice. Purification under anaerobic conditions was always accompanied by loss of activity, but this activity could be stimulated 4- to 10-fold by the addition of FMN to the reaction mixture. This (reversible) loss of activity was greatest when an oxygen scavenger such as glucose or glucose oxidase was added to the buffers. When the enzyme was purified aerobically, there was a much smaller loss of activity, but it could not be recovered by added FMN. The flavin requirement for this enzyme is discussed in detail below. As shown in Table 1, a large loss in activity occurred on the first DE-52-cellulose column. This loss occurred in both aerobic and anoxic preparations. After the work here had been completed, we found that adding Fe2+ [100 ,uM Fe(NH4)2SO4] and a thiol reducing agent (1 mM cysteine) to the buffer increased the recovery of formate dehydrogenase activity at this step from 20% to approximately 70%. These reagents appear to reconstitute the Fe/S center(s) of formate dehydrogenase, just as they did for the hydrolyase portion of the methane monooxygenase of this organism (16). The specific activity of formate dehydrogenase in crude
FIG. 3. Polyacrylamide gel electrophoresis of purified formate dehydrogenase. (A) Lanes 1 and 2, Native disk gels stained for formate dehydrogenase activity and for protein with Coomassie blue R-250, respectively. (B) Lane 3, SDS-polyacrylamide gel stained for protein with Coomassie R-250 (5 ,ug of protein added). Lane 4, Molecular mass markers: myosin (205 kDa), E. coli ,B-galactosidase (116 kDa), rabbit muscle phosphorylase B (97 kDa), bovine serum albumin (66 kDa), hen egg albumin (45 kDa), and carbonic anhydrase (29 kDa). extracts ranged from 0.12 to 0.3 ,umol of NAD+ reduced per
min per mg of protein. The purification process (Table 1) yielded near homogeneous enzyme having a specific activity of approximately 32 U/mg. Figure 3A, lane 1, is an activity stain of formate dehydrogenase on a native polyacrylamide gel. Lane 2 shows a Coomassie stain of the enzyme following purification; it appears to be greater than 90% pure. Molecular mass and subunit structure. The molecular mass of M. trichosporium formate dehydrogenase determined on a calibrated Sephacryl S-300 column (0.9 by 58 cm) was approximately 315 kilodaltons (kDa) (Fig. 4). This value is an average from values for relatively pure samples applied to the column and for a preparation that had only one previous step of purification. The latter sample was used to determine whether the two size species of formate dehydrogenase that often exist early in the purification procedure were comparable in size to the purified components. They seemed to be, as sizes of 309 and 155 kDa were measured. The 315-kDa form of formate dehydrogenase was demonstrated by electrophoresis in a 10% polyacrylamide gel in the presence of 0. 1% SDS to have two subunits (Fig. 3B, lane 3). The a-subunit had a mean molecular weight of 102,600 3,900, and the P-subunit was 54,800 + 2,800 (five determinations). Since the low-Mr form of formate dehydrogenase also showed the same subunit size pattern (data not shown), this suggests that the enzyme is an a,B heterodimer and that the high-Mr form is an (X2P2 tetramer. The physical and
TABLE 1. Purification of M. trichosporium formate dehydrogenase Purification step
Crude extract First DE-52-cellulose Second DE-52 cellulose DEAE-Sephadex A-50 First Sephacryl S-200 Second DEAE-Sephacryl A50
Activity
Vol
Protein Concn
Total
Concn
Total
Yield
Sp act
Purification
(ml)
(mg/ml)
(mg)
(U/ml)
(U)
(%)
(U/mg)
(fold)
100 18 23 12 5 1
0.25 2.2 9.7 17.7 26.6 32.3
1 8.6 39 71 106 130
490 87 14 8 5.9 2.0
2.0 1.1 2.0 1.0 0.35 0.09
980 96 28 8 2 1.8
2.3 2.3 19.4 17.7 9.2 5.8
1,153 207 272 142 54 11.6
VOL. 172, 1990
FORMATE DEHYDROGENASE FROM M. TRICHOSPORIUM
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kinetic characteristics of the enzyme are summarized in Table 2, where they are compared with formate dehydrogenases from "Pseudomonas oxalaticus" and a Moraxella species. The metal cofactor. M. trichosporium formate dehydrogenase is a brown protein which has a peak at 278 nm and a rather nondescript shoulder in the 300- to 400-nm region when isolated in the deflavo form. The addition of 5 mM sodium formate bleached the enzyme to about one-fourth the extent of dithionite (data not shown). The amounts of iron, nickel, and molybdenum have been determined on several preparations of formate dehydrogenase. It has not been practical to analyze preparations of very high activity (>30 U/mg) because of the relatively small amount of enzyme that was available. Based on their purity and metal content, we calculated the range of metal concentrations in several preparations (Table 2). Nonheme iron was the major metallic component, with values ranging between 11 and 18 g-atom per mol of enzyme (Mr 315,000). Inorganic sulfur was also present, with values between 15 and 20 mol/mol of enzyme being obtained. There were only traces of molybdenium and nickel. Selenium, which is present in this enzyme from a number of anaerobes, was not analyzed. Reversible inactivation and reconstitution of activity with FMN. When formate dehydrogenase from M. tnchosporium
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was prepared under aerobic conditions, the enzyme was fully active as isolated. However, when this enzyme was purified anaerobically, we found that these preparations lost most of their activity following an early chromatography
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FIG. 6. Inactivation and FMN-mediated reconstitution of formate dehydrogenase (FDH) activity. Formate-induced inactivation was carried out for 2 min (or in panel A for the time indicated) in a 50-pd reaction mixture in a microfuge tube containing enzyme (as indicated), (see formate, and a glucose-glucose oxidase 02 scavenger. After this preincubation, the normal formate dehydrogenase assay mixture Materials and Methods), with or without a flavin cofactor (as indicated), was added to this solution, which was then transferred to a cuvette to monitor NAD reduction. (A) Time course of inactivation by formate added at time zero (0), and the effect of 2 ,uM FMN (0) or 2 p.M FAD (A) in reconstituting this activity. Formate dehydrogenase, 19 ,ug of protein. (B) Effect of sodium formate concentration on the reversible inactivation of formate dehydrogenase. The enzyme (19 p.g of protein) was inactivated as described above with the concentration of formate indicated. After 2 min, the 50-pul preincubation mixture was added to 1 ml of assay mixture to determine the extent of inactivation. (C) Effect of FMN concentration on reactivating formate-inactivated formate dehydrogenase. A tube of formate dehydrogenase of high specific activity was inactivated for 2 min with 0.5 mM formate as described above. Samples of enzyme (1.3 jig of protein) were removed and added to the assay mixture containing FMN at the concentrations indicated. The dashed line represents the initial activity of the formate dehydrogenase, or the point at which the enzyme would have recovered 100% of its original activity.
FORMATE DEHYDROGENASE FROM M. TRICHOSPORIUM
VOL. 172, 1990
(a)
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0.1
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1 min
(b)
FIG. 7. Formate-induced inactivation of "P. oxalaticus" formate dehydrogenase and its reactivation by FMN. "P. oxalaticus"
formate dehydrogenase was obtained from Sigma Chemical Co. (St. Louis, Mo.) and used without further purification (specific activity,
