Vol. 122, No. 3 Printed in U.S.A.

JOURNAL OF BACMERIOLOGY, JUNE 1975, p. 1364-1374 Copyright 0 1975 American Society for Microbiology

Oxidation of C1 Compounds by Particulate Fractions from Methylococcus capsulatus: Properties of Methanol Oxidase and Methanol Dehydrogenase ANN M. WADZINSKI AND DOUGLAS W. RIBBONS* Department of Biochemistry, University of Miami School of Medicine, Miami, Florida 33152

Received for publication 27 December 1974

Methanol (and formaldehyde) oxidizing activities in crude extracts of Methylococcus capsulatus are associated mainly with particulate fractions sedimenting between 3,000 and 40,000 x g. Most of the phenazine methosulfate (PMS)-dependent methanol (and formaldehyde) dehydrogenase activity observed resides in the soluble fraction but represents only 40% of the total (PMS dependent plus independent) activity. Both PMS-dependent methanol dehydrogenase activity and PMS-independent methanol oxidase activity are found in particulate fractions, and the PMS-dependent dehydrogenase is easily solubilized by treatment with certain phospholipases or detergents. The properties of the PMS-dependent dehydrogenase activities in the soluble fraction and that solubilized from the particles suggested that they may be identical proteins. Their pH optima, temperature dependence, thermolabilities, and sensitivities to the presence of specific antisera were indistinguishable. Homogeneous preparations of the enzyme proteins obtained from the soluble fractions of extracts and the particulate fractions solubilized by detergents had similar: (i) electrophoretic mobilities in native and denatured states (subunit size in sodium dodecyl sulfate 62,000 daltons); (ii) molecular radii under native conditions, (iii) visible absorption spectra, Xmax 350 nm, (iv) kinetic constants for methanol and formaldehyde; (v) substrate specificity; and (vi) immunological characteristics-antisera to each enzyme preparation showed precipitin lines of identity to either of the enzymes. It is suggested that the major site of methanol and formaldehyde oxidation in M. capsulatus occurs on the intracytoplasmic membranes in vivo and is coupled to oxygen reduction. 1

The oxidation of methanol by enzymes extracted from microorganisms has been the subject of numerous investigations (for reviews see references 22 and 24). A variety of soluble methanol oxidizing enzymes have been isolated from both obligate and nonobligate methyltrophs (1-6, 13, 14, 16, 19-21, 25). The two systems, which have been well characterized, are: (i) the pteridin methanol dehydrogenases first obtained from Pseudomonas M27 by Anthony and Zatman (1-5) and later from the obligate methylotroph Methylococcus capsulatus (19-21) in pure form; and (ii) the flavoprotein methanol oxidases from yeasts (13, 25). The quantitative importance of these activities has not always been clear except in the case of mutants of Pseudomonas AM1 which lack methanol dehydrogenase activity and are unable to grow on methanol (15). The pteridin methanol dehydrogenases obtained from bacteria can only be assayed in the

presence of phenazine methosulfate (PMS), an auxilliary electron carrier required for the nonenzymic reduction of 2,6-dichlorophenolindophenol blue or molecular 02; the natural electron acceptors for these enzymes are unknown. Enyzmic activity is markedly stimulated by the presence of NH,+ salts, this being attributed to NH. itself (2, 19). Very little is known about the chemical nature of the pteridin in methanol dehydrogenase since it is very photochemically labile and sufficient quantities of it have not been obtained for chemical characterization. However, recent work (G. T. Sperl, Ph.D. thesis, University of Texas, 1973) indicates that it is of lumazine type. The role of the pteridin cofactor in methanol oxidation remains unclear at this time. While studying the ability of particulate fractions from cell-free extracts of M. capsulatus to oxidize methane we observed that methanol and formaldehyde readily stimulated

1364

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METHANOL OXIDASE AND DEHYDROGENASE

respiration in the absence of auxilliary electron carriers (23). In view of the extensive intracytoplasmic membrane systems found in M. capsulatus (10, 24, 27), the particulate methanol and formaldehyde oxidizing activity warranted further investigation, and this is the subject of this paper. MATERIALS AND METHODS Organism and fractionation of extracts. M. capsulatus was maintained, cultivated, and monitored for purity as described previously (23). Extracts of cultures growing exponentially on methane as sole source of carbon were obtained by liquid shear in a French press and fractionated by differential centrifugation (23). Assay of oxidase and dehydrogenase activities. Oxidase and dehydrogenase activities of extracts of M. capsulatus were assayed polarographically with a Clark oxygen electrode. Oxidases and dehydrogenases are distinguished as the activities observed in the absence and presence of phenazine methosulfate (PMS)-definitions of the activities observed and their distinction are given below. Usually each assay mixture for oxidase activity contained: 15 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 9 (2.9 ml); particle or soluble fraction (0.1 ml); and substrate (reduced nicotinamide adenine dinucleotide phosphate [NADH], methanol or formaldehyde) as indicated. For the dehydrogenase assays, reaction mixtures were supplemented with phenazine methosulfate (330 nmol). Solubilization of methanol oxidizing activities from particle fractions. The particulate methanol oxidizing activity was solubilized in the following ways. Particles were incubated at 37 C for 1 h with phospholipase A from Vipera russelli at pH 6.5, phospholipase C from Clostridium welchii at pH 7.3, or phospholipase D from cabbage (Sigma) at pH 5.6. Controls lacking the phospholipases were conducted at the various pH values. Particles were also incubated on ice at pH 7.1 for 30 min with 0.1% G-2090 (polyoxyethylene sorbitol oleate-polyoxyethylene amine blend)-0.1% Span 80 (sorbitan monooleate)-0.1% Tween 80 (polysorbate 90 USP). The detergents were obtained from Atlas Chemical Industries, Inc. Methanol oxidase activity and methanol dehydrogenase activity were measured on samples taken periodically during the incubation after centrifugation at 40,000 x g for 10 min. Purification of soluble and solubilized methanol dehydrogenase. Methanol dehydrogenase activity from crude soluble extracts (40,000 x g supernatants) and that solubilized from the particle fractions by treatment with the detergent G-2090 were both purified by sequential fractionation on DEAE-cellulose and Sephadex as described by Patel et al. (21), with the following modifications. The soluble crude extract and the supernatant from G-2090-treated particles were applied to a DE-52 (DEAE-cellulose) column equilibrated with 20 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 8.0) containing 25 mM methanol. The methanol dehydrogenase activities

1365

were eluted with the equilibration buffer without a gradient. Combined fractions (100 ml) with the highest specific activities were concentrated to 15 ml by ultrafiltration through an Amicon PM-10 membrane (Amicon Corp., Lexington, Mass), and applied to Sephadex G-200 columns which were eluted with the same buffer. The peak fractions gave single protein bands on gel electrophoresis. Analytical procedures. Formaldehyde solutions were prepared by heating an aqueous solution of a weighed amount of paraformaldehyde in a water bath for 1 h. Formaldehyde was determined by the chromotropic acid method of Friscell and MacKenzie (12). Formate was determined with the NAD-dependent formate dehydrogenase found in the supernatant fractions of M. capsulatus. These assays were performed immediately after methanol (or formaldehyde) oxidations were complete in an 02-absorbance cuvette (23). Reaction mixtures contained: KH2PO4NaOH buffer (pH 8.0; 45 ,mol), methanol (625 nmol) or formaldehyde (1,250 nmol), particle fraction (0.1 ml), and soluble fraction (0.05 ml), in a total volume of 2.5 ml. After oxygen consumption had ceased, NAD (2.5 Amol) was added, and the change in adsorbance at 340 nm was recorded. Suitable controls in the absence of the soluble fraction, and with authentic formate solutions, were included. Protein was measured by the method of Lowry et al. (18) and by absorbance at 280 nm for column effluents. Determination of kinetic constants. The Km values for methanol and formaldehyde oxidations were determined from polarographic determinations of initial reaction rates and computed using the program Woenzyme based on the method of Bliss and James (7) to give 95% confidence limits of the determinations. Disc gel electrophoresis. The article by Cooksey (8) was used as a general reference for the technique of disc gel electrophoresis. Legends to the figures detail the individual experiments. The activity stain consisted of 980 Mmol of tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 9.0), 80 mg of nitroblue tetrazolium (Sigma), 1.4 mg of phenazine methosulfate (Sigma), and 3 mmol of methanol, in a total volume of 100 ml. The gels were incubated in the dark at room temperature in this solution for 1 h. Immunological methods. Antisera were prepared by Ron Menichino through the courtesy of Frank de Velasco, Dade Division of American Hospital Supply, using New Zealand white rabbits. The rabbits were injected twice with crude 40,000 x g supernatant or supernatant from particles treated with G-2090 containing 2 mg of protein in 1 ml of Freund complete adjuvant. The injection interval was 2 weeks, and the rabbits were bled 1 week after the second injection. The antiserum to the crude 40,000 x g supernatant is abbreviated anti-S antiserum, and the antiserum to the supernatant solubilized from particles treated with G-2090 is abbreviated anti-PS antiserum. Ouchterlony double-diffusion plates were prepared by placing 2 ml of 0.5% Ionagar no. 2 (Oxoid) in saline-phosphate (0.05 M) buffer (pH 7.5) in Falcon disposable petri dishes (50 by 12 mm; Scientific

1366

WADZINSKI AND RIBBONS

J. BACTzRioL.

Products). The wells (3 mm in diameter) were made using an immunodiffusion punch set (LKB Instruments, Inc.). The dilution of antigen which gave the sharpest bands was determined in preliminary tests. The concentration of protein for the purified soluble enzyme was 37 ug/ml and for the purified solubilized enzyme was 56 Ag/ml. All immunodiffusion reactions were incubated for 48 h at room temperature in a humid atmosphere. Defimitions of methanol oxidizing activities. Methanol oxidizing activities found in cell extracts of M. capsulatus have been categorized on the basis of both assay procedure and their distribution in sedimentable fractions. The following definitions will be used throughout. Particulate methanol oxidase (PO) is used to describe the activity in particulate fractions that is independent of PMS. Particulate methanol dehydrogenase (Pdh): the addition of PMS to PO assays results in a further stimulation of oxygen consumption. The difference between the total rate of 0° consumption (+PMS) and that due to PO (-PMS) represents Pdh activity. It is not clear what the PMS assay is measuring, and so the Pdh activity is an operational definition. Soluble methanol dehydrogenase (Sdh) is the total rate of 02 consumption observed when methanol is oxidized by 40,000 x g supernatants in the presence of PMS. Although these supematants contain small amounts of the oxidase activity (-PMS), this represented 5 to 15% of activity in the soluble fraction, and it has been ignored in the results presented. Solubilized particulate methanol dehydrogenase (PSdh) is the PMS-dependent methanol dehydrogenase activity that is solubilized from the particulate fractions after treatment with phospholipases or detergents. PMS-independent activities were not observed.

genase activities when formaldehyde is supplied as the substrate, which is strikingly similar to the results for methanol (Table 1). Products and stoichiometry of methanol and formaldehyde oxidation catalyzed by particles. Formate was the only product found after methanol and formaldehyde had been oxidized by the particulate oxidase(s). Carbon dioxide was not formed unless the soluble fraction, supplemented with NAD, was also present. This gave a low yield of carbon dioxide (60% of the theoretical expected), similar to the recoveries obtained when standard solutions of formate were used as controls. Formate was detected as the product of methanol and formaldehyde oxidations by reaction with thiobarbiturate. Using the spectrophotometric assay for formate, (formate dehydrogenase plus NAD), equimolar quantities of NADH (and thus formate) were formed from both methanol and formaldehyde (and standard formate solutions). Th>amount of oxygen consumed is cohsistent -with the quantitative oxidation of both substrates to formate (Table 3), and one proton equivalent is released per mole of methanol or formaldehyde supplied. These data and the release of one proton equivalent (Table 3) are consistent with the following equations for methanol and formaldehyde oxidation by the particulate fractions: HCOO- +H+ +H,0 CH,OH+O,, 02 -HCHO + O/202 -- eHCOO- + H+

RESULTS

TABLE 1. Distribution of methanol oxidizing activities in extracts of M. capsulatus

Distribution of methanol oxidizing activities in extracts of M. capsulatus. PO activities are evenly divided between 3,000 to 10,000 x g (P2) and 10,000 to 40,000 x g (P3) pellets (fractionation scheme is given in reference 23). Small amounts of methanol oxidase activity (distributionally called PO activity) could be demonstrated in the 40,000 x g supematant. Conversely, the methanol dehydrogenase (Pdh and Sdh) activities reside mainly in the supernatant fraction (Table 1). The specific activities (nmol of 0, consumed/min/mg of protein) in the cellular fractions were: P2 (PO plus Pdh), 87.5; P. (PO plus Pdh), 93.1; S (Sdh), 10.5. These values are the average of five different preparations. Distribution of formaldehyde oxidizing activities in extracts of M. capsulatus. It was also observed that formaldehyde could be oxidized by the particulate fractions in the absence of PMS, and, as for methanol as substrate, this activity was stimulated further by PMS. Table 2 shows the distribution of oxidase to dehydro-

96 Distribution in cell fractions Activity

PO Pdh or Sdh PO + Pdh

s

P2

P3

42.4

41.2

16.4a

18.2 30

18.2 29.4

63.6b 40.60

aOxidase activity found in the soluble fraction. bSdh activity only. TABLE 2. Distribution of formaldehyde oxidizing activities in extracts of M. capsulatus % Distribution in cell fractions

Activity

PO Pdh or Sdh PO + Pdh

P2

Ps

S

44.2 15.5

43.2 20.7 29.6

63.8b

29.4

12.6a

41.01

a Oxidase activity found in the soluble fraction. 0 Sdh activity only.

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1367

METHANOL OXIDASE AND DEHYDROGENASE

Solubilization of methanol oxidizing activity from the particles. The existence of both methanol oxidase and methanol dehydrogenase (PMS linked) activities on the particles, and also the presence of a methanol dehydrogenase in the soluble fraction of M. capsulatus, suggested that the proteins catalyzing the primary dehydrogenation of the substrate might be identical, and that the soluble dehydrogenase activity, for which there is no known natural electron acceptor, is recovered as a result of its easy dissociation from the membrane fractions of the cell, either by disintegration and fractionation of the cells or as a physiological consequence of using older cells. The latter idea is supported by the observation that when the pH value of cultures of M. capsulatus rises above 7.0 there is a decrease in the specific and total activities of the methanol oxidase and an increase in the methanol dehydrogenase activity. If the pH

value-of'the cultures is maintained between 6.8 and 7.0, there is little loss of oxidase activity even during stationary phase (unpublished observations). Attempts to solubilize the methanol oxidase activity with phospholipases and detergents completely destroyed the oxidase activity, but gave soluble preparations that would oxidize methanol (and formaldehyde) only when the artificial electron acceptor, PMS, was present, i.e., methanol dehydrogenase activity had been solubilized from the particles, with concomitant loss of oxidase activity. Phospholipase A and phospholipase D, but not phospholipase C, were effective in solubilizing a methanol dehydrogenase activity (PSdh) from the particles (Table 4). Treatment of the particles with a detergent G-2090 (an anionic-cationic blend) gave similar results, but Span 80 and Tween 80 gave poor recoveries of

TABLE 3. Stoichiometry of methanol and formaldehyde oxidations by particles of M. capsulatus

TABLE5. Solubilization of methanol oxidizing activity with detergents

Substrate supplied

Methanol Methanol Methanol

Formaldehyde

Amt

250 nmol 625 nmol 30 gmol

02 HCOOH+ consumed formed formeda

243 531 ND

ND, 525 ND

ND ND 32

266 nmol

134

ND

ND

1,250 nmol

512

1024

ND

aProton release was measured in a volume of 3 ml containing tris(hydroxymethy)aminomethane-hydrochloride (pH 9) (50 gmol), particulate fraction, and methanol (30 umol) by automatic titration with 0.14 N NaOH using a Radiometer (model TTT1,) pH stat. 'ND, Not determined.

Total act (nmol of OJ/min) Treatment Supernatant

None G-2090 Span 80 Tween 80

Pellet

+PMS

+PMS

-PMS

9,200 103 103

12,000 240 6,320 6,460

8,260 0 0 0

a Particles from Methylococcus capsulatus were incubated on ice at pH 9.0 with 0.1% detergent for 5 h. The particles were centrifuged at 25,000 x g for 15 min and resuspended. Both the particles and the supernatant were tested for methanol oxidase and methanol dehydrogenase activity with an oxygen electrode as described.

TABLE 4. Solubilization of methanol oxidizing activity with phospholipasesa Fraction and treatment

Particles

Supernatant from phospholipase A-treated particles Supernatant from phospholipase D-treated particles Supernatant from phospholipase C-treated particles Particles after phospholipase A treatment Particles after phospholipase D treatment Particles after phospholipase C treatment

Sp act (nmol of O/min/mg of protein)

Total act (nmol of O/min)

-PMS

+PMS

-PMS

+PMS

6.9 0 0 0 0 0 12.6

11.5 22.0 11.4 0 0 0 15.3

331 0 0 0 0 0 302

554 244 182 0 0 0 366

aParticles (3.0 ml, 12 mg of protein/ml) from M. capsulatus were incubated for 30 min with the various phospholipases at 37 C. The phospholipase A (0.25 mg) incubation was done at pH 6.5, phospholipase C (0.5 mg) at pH 7.3, and phospholipase D (0.1 mg) at pH 5.6. The oxidase activity was measured with an oxygen electrode, and the dehydrogenase was measured manometrically as described.

J. BAcTERioL.

WADZINSKI AND REBBONS

1368 540 520

500 140

(i) Thermal stability. Crude soluble (Sdh) and solubilized (PSdh) preparations gave similar inactivation rates when held at 45, 60, 75, 85, or 100 C for various periods of time. Figures 1 and 2 show the time courses of inactivation at several temperatures. The particulate methanol activity (P0) was very sensitive to elevated temperatures, with almost all activity being lost within 20 min at 45 C and before 15 min at 60 C. The loss of oxidase activity in the particles is probably due to disruption of the methatransport system, since the loss of totalelectron \P dh nol oxidizing activity the and particles plus to theofSdh PSdh(P0 was similar k\Pdh) profiles. dli Consistent with this view is that loss of metha\ Sdh nol oxidase activity results in an apparent increase in the particulate dehydrogenase activity (Pdh) (Fig. 1 and 2). \ dh (ii) Effect of temperature on activity. The \ \\ \1 PARTICULATE various methanol oxidizing activities have simi\ TOTALACTIVITY -

-

2t

> 120 F

< _100 oo '< 80 z 80 O

60

40 20

P -OXIDASE

-

10

20

100

a

a

I

30

40

50

TOTAL PARTICULATE

60

ACTIVITY, 75°C

70

TIME (minutes) FIG. 1. Temperature stability of the methanol oxidizing activities of M. capsulatus at 60 C. The Pdh, 750C \ dh, 750C \ preparations were held at 60 C, and aliquots were removed and assayed with an oxygen electrode as >/ described. The initial specific activities (nmol of u\ 750C \ O,min/mg of protein) were: P-oxidase, 8.4; Pdh, 2.3; < 50 total particulate activity, 10.7. PSdh (crude solubi- < \ lized methanol dehydrogenase), 5.9; and Sdh (soluble ~ methanol dehydrogenase), 3.1. P-oxidase, Pdh, and Z \ S75dh,0C total particulate activity are from the same preparation. Total particulate activity refers to the additive 25 ALL ACTIVITIES. activities observed in particulate fractions, namely 350 & 1000C PO plus Pdh. 75

>_

the solubilized dehydrogenase and completely destroyed PO activity (Table 5). Suspensions of the particles in buffer without these reagents did not significantly solubilize the methanol dehydrogenase (Tables 4 and 5). Comparative properties of crude-soluble (Sdh), crude-solubilized (PSdh), and particulate preparations of methanol dehydrogenase (Pdh) and methanol oxidase (PO). The effects of temperature, pH, and specific antisera on the various methanol oxidizing activities in the fractions were compared in an attempt to demonstrate the similarity, or not, of the soluble and solubilized methanol dehydrogenases, asc as well as toto compare the activity when associas T heP nd Pdh P0 and ated wela with the particles. The activities were also included in this survey,

comparethe.

P -OXIDASE,

750C

.10

15

TIME (minutes) FIG. 2. Temperature stability of the methanol oxidizing activities of M. capsulatus at 75,85, and 100 C.

The preparations were held at the various temperatures, and aliquots were removed and assayed with an oxygen electrode as described. The initial specific activities (nmol of 0,min/mg of protein) were: P-oxidase, 8.4; Pdh, 2.3; total particulate activity, 10.7; Pdh, 2.3; PSdh (crude solubilized methanol dehydrogenase), 5.9; and Sdh (crude soluble methanol dehy2.6. P-oxidase, Pdh, and the total particudrogenase), late activity are from the same preparation. The legend to fig. 1 gives the meaning of total particulate activity.

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METHANOL OXIDASE AND DEHYDROGENASE

lar activity-temperature profiles (Fig. 3), with all increasing more than twofold when the temperature is raised from 28 to 55 C. (iii) Effect of pH on activity. The pH optima for methanol oxidation was the same for the particulate oxidase and the dehydrogenase. The soluble and solubilized dehydrogenase preparations gave similar profiles. The pH optima are about 9.5 (Fig. 4), but particulate activities are more active at lower pH values, possibly reflecting the influence of association of the proteins on the membranes. (iv) Effect of antisera on the soluble and solubilized methanol dehydrogenases. Antisera prepared against crude soluble and solubilized methanol dehydrogenase preparations from M. capsulatus, and against pure methanol

1369

dehydrogenase from Hyphomicrobium sp. inhibited the particulate oxidase and dehydrogenase activities to the same degree (about 25 to 35%), but this was less than the inhibition of the soluble and solubilized methanol dehydrogenase preparations (40 to 55%) (Table 6). Association of methanol oxidizing activity with the particulate fractions apparently confers some degree of resistance to inhibition by the specific antisera. Antisera prepared for unrelated purposes did not significantly inhibit methanol oxidation catalyzed by the pure enzymes or particulate fractions. Purification of the soluble and solubilized methanol dehydrogenases from M. capsulatus. Homogeneous preparations of

200 TOTAL PARTICULATE ACTIVITY

160 0._ 0

11I.-

E -

u 120

E

I I

0

x

0

E

80

I

F

01-10

> I--

40

6

30

40 50 TEMPERATURE (°C)

7

8

60

FIG. 3. Methanol oxidizing activity of M. capsulatus versus temperature. Oxygen uptake was measured with an oxygen electrode as described. PSdh is the crude solubilized methanol dehydrogenase, and Sdh is the crude soluble methanol dehydrogenase.

9

10

pH

FIG. 4. Effect of pH on methanol oxidizing activity of M. capsulatus. Activity was measured with an oxygen electrode as described. The maximum specific activities (nmol of 02/min/mg of protein) were: P-oxidase, 9.6; Pdh, 2.6; PSdh (crude solubilized methanol dehydrogenase), 4.9; and Sdh (crude soluble methanol dehydrogenase), 2.6; at pH 9.5.

TABLE 6. Inhibition of methanol oxidizing activities by antisera Antiserum Antiserum Antiserum (mg of protein)

Anti-Hyphomicrobium methanol dehydrogenase antibody Anti-soluble methanol dehydrogenase antibody Anti-solubilized methanol dehydrogenase antibody

9.0 4.1 4.5

P0~%PdHInhibition Sdh

PSdh

28 35 34

41 56 55

26 29 29

44 53 49

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J. BACTERIOL.

WADZINSKI AND RIBBONS

methanol dehydrogenase were obtained from the particulate (PSdh) and soluble fractions (Sdh) of M. capsulatus. The purifications are summarized in Table 7 and detailed above. Fractions obtained after Sephadex G-200 chromatography had similar specific activities and gave single bands on disc gel electrophoresis after staining for protein or methanol dehydrogenase activity with tetrazolium salts as elec-

95% confidence limits of the determinations were also much broader. The Km values for methanol and formaldehyde for the various preparations are given in Table 8. The pure soluble and solubilized dehydrogenases catalyze the oxidation of several primary alcohols, formaldehyde, and to a lesser extent

tron acceptors.

Physical properties of purified particulate and soluble methanol dehydrogenase. The two enzyme preparations migrated as single bands with identical electrophoretic mobility in polyacrylamide gels (Fig. 5). Similar results were obtained in sodium dodecyl sulfate SDSgels, and molecular weight values of 62,000 were obtained.(Fig. 6). The molecular radii of both dehydrogenases also appear to be the same since the enzymes elute from Sephadex G-200 in the same fractions (Fig. 7), and these correspond to molecular sizes of about 120,000 daltons (not determined accurately). Both protein preparations were pale yellow and showed absorption maxima at 280 and 350 nm (Fig. 8). These spectra are similar to those published for methanol dehydrogenase by Anthony and Zatman (4, 5) for Ps.M27 and by Patel et al. (19, 21) for M. capsulatus (soluble). The shoulder between 400 and 450 nm is typical but variable and could be due to the breakdown of the chromophore in the enzyme, since the protein-free cofactor is known to be very photolabile (G. T. Sperl, Ph.D. thesis, University of Texas, 1973). Catalytic properties of the purified particulate and soluble methanol dehydrogenases. The purified soluble methanol dehydrogenase (Sdh) and solubilized methanol dehydrogenase (PSdh) and the particulate methanol oxidase (PO) have approximately the same Km values for methanol (Fig. 9). The 95% confidence limits for each of these activities coincide to a large extent. The Km values obtained for formaldehyde as substrate varied to a greater extent; the

Sdh' PSdh

FIG. 5.

Polyacrylamide gel electrophoresis of the

soluble and solublized methanol dehydrogenases. The elect rophoresis

was

done

by the method of Davis (9).

TABLE 7. The purification of soluble and solubilized methanol dehydrogenases Fraction

Soluble

Solubilized

Sp act Fold (nmol/min/mg purification of protein)

Purification step

Total act (nmoVlmin)

Recovery M

(mg/ml)

Crude supernatant DEAE-cellulose Sephadex G-200

31,700 17,900 4,850

100 57 15

29.4 15.2 2.4

4.0 23.6

1.0 5.9

67.3

16.8

Crude supernatant DEAE-cellulose Sephadex G-200

7,150 4,620

100 65 13

11 4.8 0.9

6.5 49.8 69.1

1.0 7.6 10.6

964

Protein

VOL. 122, 1975

METHANOL OXIDASE AND DEHYDROGENASE

1371

acetaldehyde (Table 9). The ratios of the activi- hydrogenases gave precipitin lines of identity ties for these substrates are similar and closely to antisera prepared against either preparation parallel the spectrum of substrates oxidized by in Ouchterlony double diffusion plates (Fig. 10). the particulate methanol oxidase, which is inDISCUSSION cluded in Table 9 for comparison. Of the secondary and tertiary alcohols tested, namely, isoThe ubiquitous occurrence of intracytoplaspropanol, iso-butanol, sec-butanol, tert-buta- mic membrane systems in obligate methylonol, and iso-pentanol, none were substrates. trophs suggests that they support physiological Serological properties of the purified par- and biochemical activities important, and posticulate and soluble methanol dehydrogen- sibly unique, for the growth and survival of this ases. Antisera prepared against the crude enzyme solubilized from the particles treated with G-2090 and the crude soluble fraction of M. capsulatus had both previously been shown to inhibit unpurified soluble and solubilized methPSdh 60 anol dehydrogenase activities (Table 6). The purified soluble and solubilized methanol deE

-a 200

\.

40

ALDOLASE

8 I.

3

E

100

c

S dhANDQ PSdh

50

\&S dh

\

*

OVALBUMIN

wY

20

D

\ CHYMOTRYPSINOGEN *

MYOGLOBIN

RIBONUCLEASE'

20

40

60

CYTOCHROME

8

1

80

100

RELATIVE MIGRATION

(ORIGIN)

F

u

C

L-

(l

FIG. 6. Molecular weight determination of the subunits of the soluble and solubilized n-ethanol dehydrogenases. The molecular weights were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the method of Shapiro and Vinuela (26).

io -

-

I

ANODE)

~

I

I

I

l

40 80 120 160 200 ELUTION VOLUME (ml) FIG. 7. Elution patterns of soluble and solubilized methanol dehydrogenases from Sephadex G-200. Assays were done with an oxygen electrode as described.

A

2

03

02

200

250

300

350

400

450

500

550

600

WAVELENGTH (nm)

FIG. 8. Spectra of the purified soluble methanol dehydrogenase and solubilized methanol dehydrogenase. The protein concentrations (mg/ml) of the soluble methanol dehydrogenase samples were S,, 0.12; and S2, 2.4; and the solubilized samples were PS1, 0.09; and PS2, 0.9.

1372

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WADZINSKI AND RIBBONS

group of bacteria. The association of the activities of the primary reaction of formaldehyde fixation into a sugar phosphate with particulate fractions of M. capsulatus lends support to this notion (22). Our own studies have centered around the role of these membranes in the oxidation of Cl compounds. Thus, we have observed that methane, methanol, and formaldehyde are all oxidized by particle preparations from M. capsulatus, with concomitant reduction of oxygen. Only methane oxidation requires NADH as an external electron donor (23); methanol and formaldehyde are oxidized quantitatively to formate, a product also formed

when methane is provided as a substrate in the presence of NADH (23). Previous descriptions of methanol oxidizing activities in M. capsulatus have shown that a soluble dehydrogenase is readily obtained (19-21) and is similar to dehydrogenases prepared from nonobligate methylotrophs (1-5, 19-21). This soluble dehydrogenase, which we also observed, has only been assayed in the presence of PMS; the natural electron carrier is not known. Particle preparations of M. TABLE 8. Kinetic constants for methanol and formaldehyde Km (uM)

95%

Meh-Fra-Confi'dence limits

Enzyme

nol

Soluble methanol dehydrogenase Solubilized methanol dehydrogenase Particulate methanol oxidase

dehyde

12-27

19 71

30-130 10-26

48

11-105 12-23 46-89

17 18

66

TABLE 9. Substrate specificity of the particulate methanol oxidase, the soluble methanol dehydrogenase, and the solubilized methanol dehydrogenase % Activity with methanol as substrate

Substrate

-0.1

0

0.1

0.2

METHANOL FIG. 9. Determination of Km values for methanol; the kinetic studies were done as described. A

7

Methanol Ethanol n-Propanol n-Butanol

Formaldehyde

Acetaldehyde

B

I

3

7

PO

Sdh

PSdh

100 93 48 56 76 9

100 89 46 43 72 7

100 90 54 49 74 10

i

:3

FIG. 10. Immunodiffusion of soluble and solubilized methanol dehydrogenase. Wells 1, 3, 5 and 7 contained solubilized methanol dehydrogenases; wells 2, 4, 6 and 8 contained soluble methanol dehydrogenase. The center well of A contained anti-PS antiserum and B contained anti-S antiserum.

VOL. 122t 1975

METHANOL OXIDASE AND DEHYDROGENASE

capsulatus which oxidize methanol (and formaldehyde) to formate show higher activities in the presence of PMS. It seemed possible then that the oxidase activities observed on the particles may be physiologically and quantitatively important for the oxidation of methanol and formaldehyde. Accordingly, a study of this system was undertaken and compared to the methanol oxidizing activities found in soluble fractions of cell extracts. When the particles are treated with phospholipase A or C, or the anionic-cationic detergent G-2090, methanol oxidase activity is completely lost; however, proteins solubilized by these procedures have the ability to oxidize methanol (and formaldehyde) when PMS is also provided. Thus it seemed possible that the soluble dehydrogenase found in M. capsulatus might be associated in vivo with the membranes in M. capsulatus, but that-the disintegration and fractionation procedures used allow a portion to appear as a soluble protein in extracts of cells. Membrane fractions of M. capsulatus also contain the PMS dehydrogenase activity characteristic of the soluble fraction. Its quantification relative to oxidase activity, however, is difficult to realize since it is not known if PMS short circuits electron flow in the oxidase system or represents another activity of the particulate fractions. Nonetheless the majority of the methanol (and formaldehyde) oxidizing activities observed in extracts of M. capsulatus appears to reside in the particulate fractions obtained and account minimally for about 60% of the total activities observed (Tables 1 and 2). The loss of oxidase activity and recovery of dehydrogenase activity when particles are treated with phospholipase or the G-2090 detergent (Tables 4 and 5), and also by exposure to elevated temperatures (Figures 1 and 2), provided the basis for the present report. Thus the solubilized dehydrogenase (PSdh) and that obtained from the soluble fractions (Sdh) of M. capsulatus were purified to apparent homogeneity and could not be distinguished by several physical, catalytic, and serological properties. These results, together with the quantitative distribution of the methane (23), methanol, and formaldehyde oxidizing activities, suggest that the intracytoplasmic membrane system of M. capsulatus is responsible for the oxidation of methane to formate in vivo. The copurification of the particulate methanol dehydrogenase and formaldehyde dehydrogenase activities, as well as their distribution in crude extracts, indicates that the same enzyme is responsible for both activities, a conclusion drawn earlier for the soluble methanol dehydrogenase from M. cap-

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sulatus (19-21); formaldehyde (hydrate) appears to be a substrate analogue of methanol. From the results obtained it also appears that the association of the methanol oxidizing activity with the particulate fractions confers a protection towards inhibition by antisera prepared against crude preparations of both Sdh and PSdh. ACKNOWLEDGMENTS We are grateful to John L. Michalover for help with the cultivation of M. capsulatus. We are grateful to G. T. Sperl for the anti-Hyphomicrobium methanol dehydrogenase antisera.

This work was supported by the National Science Foundation, grant no. GBO40712. D. W. R. was a Howard Hughes Medical Institute Investigator during the initial phases of this work and A. M. W. was a recipient of a Public Health Service predoctoral traineeship (NIH 1 Tnl GM02011) from the National Institute of General Medical Sciences.

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J. BACTERIOL.

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