10
Vol. 169, No. 2 JOURNAL OF BACTERIOLOGY, Feb. 1987, p. 718-727 0021-9193/87/0208718-10$02.00/0 Copyright © 1987, American Society for Microbiology Electron Microscopy of Nickel-Containing Methanogenic Enzymes: Methyl Reductase and F420-Reducing Hydrogenase LAWRENCE P. WACKETT,' ERIKA A. HARTWIEG,2 JONATHAN A. KING,2 WILLIAM H. ORME-JOHNSON, 1 AND CHRISTOPHER T. WALSH'l2* Departments of Chemistry' and Biology,2 Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received 18 July 1986/Accepted 30 October 1986 Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Two of the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducing hydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes from Methanobacterium thermoautotrophicum AH have been determined by electron microstopy. By negative stain techniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel 4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, inditating a holoenzyme molecular weight of 8.0 x 105. Methyl reductase appeared to be an oligomeric complex of dimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunit composition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methyl reductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highly concentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecular assemblies may have relevance in vivo in the construction of multiprotein arrays that function in methane biogenesis. Methanobacterium thermoautotrophicum AH is a thermo- philic member of the archaebacteria, a group thought to have diverged early in evolution from the more commonly studied eubacteria (8). This bacterium meets all of its energy require- ments by oxidation of hydrogen gas (28). Furthermore, M. thermoautotrophicum utilizes carbon dioxide as both an electron acceptor and a carbon source, producing methane and less-reduced one-carbon fragments which get incorpo- rated into cellular macromolecules as follows: 90% 4H2 + CO2 CH4 T + 2H20 10% Cell Carbon The unique CO2 reduction pathway is at the core of meth- anogenic biochemistry. It is possible that the methariogenic pathway has a long evolutionary history in this ancient group of organisms. In this context, the intracellular structure and organization of the proteins catalyzing these reactions are of great interest. M. thermoautotrophicum AH possesses several unustal cofactors and coenzymes (26), as well as four novel nickel- containing enzymes. Of these enzymes, two function as hydrogenases (12), and the third, methyl coenzyme M reductase (2, 10), catalyzes the ultimate methane-yielding reaction. The fourth nickel enzyme, CO dehydrogenase, is central in assimilation and dissimilation of acetate and two one-carbon fragments at the methyl and carbon monoxide oxidation states (24). The two hydrogenases have been shown to be distinct proteins by purification of each to hotnogeneity and by immunological methods (J. A. Fox, D. J. Livingston, W. H. Orme-Johnson, and C. T. Walsh, submitted for publication; L. Jordan, Ph.D. thesis, Massa- chusetts Institute of Technology, Cambridge, 1985). One hydrogenase reduces the methanogen deazaflavin redox * Corresponding author. coenzyme, F420, and methyl viologen, whereas the other hydrogenase reduces only methyl viologen. The properties of the bettet-characterized F420-reducing hydrogenase are summarized in Table 1. Clearly, this protein plays a signifi- cant role in methanogenesis, as evidenced by its concentra- tion in the cell, its large size, and its efficiency as a hydrogen oxidation and F420 reduction catalyst and by inhibition of methanogenesis in crude extracts by anti-F420 hydrogenase antibody (D. J. Livingston, L. P. Wackett, and C. T. Walsh, unpublished data). Another protein crucial to methanogene- sis is methyl coenzyme M reductase, which catalyzes the last step in the CO2 reduction pathway as follows: CH3S/-SO3 + 2e + 2H+ -* CH4O + HS,--SO3 The subunit structure (a202y2) of this major soluble protein (7) and the structure of its Ni tetrahydrocorphin cofactor, F430 (18, 19), have been elucidated (Table 1). Despite the purification of these nickel-containing meth- anogen catalysts, much remains unknown with respect to in vitro reconstitution of methanogenesis and the coupling of CO2 reduction with energy-conserving reactions in vivo. These deficiencies are apparent with the observation that cell disruption of M. thermoautotrophicum AH reduces methanogenesis from the whole-cell rate of 7,000 nmol/m'in per mg of protein to the maximum observed rate of 20 nmol/min per mg of protein in cell lysates (5). Possible explanations for this dramatic diminution in methanogenic rates, as discussed by Daniels et al. (5), involve destruction of a membrane or related vectorial processes, disruption of requisite in vitro protein-protein interactions, or dilution of necessary cofactors. The participation of internal tnem- branes in methanogenesis was invoked by Kell et al. (14) in the methanochondrion hypothesis. However, Sprott et al. (21) demonstrated methanogenesis in the absence of internal membranes, leaving the role of internal membrane-requiring processes questionable. The issue of potential protein- protein interactions between methanogenic enzymes has, to date, remained unexplored. In this context, the structure and 718 on July 18, 2020 by guest http://jb.asm.org/ Downloaded from

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Vol. 169, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1987, p. 718-7270021-9193/87/0208718-10$02.00/0Copyright © 1987, American Society for Microbiology

Electron Microscopy of Nickel-Containing Methanogenic Enzymes:Methyl Reductase and F420-Reducing Hydrogenase

LAWRENCE P. WACKETT,' ERIKA A. HARTWIEG,2 JONATHAN A. KING,2 WILLIAM H. ORME-JOHNSON, 1

AND CHRISTOPHER T. WALSH'l2*Departments of Chemistry' and Biology,2 Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 18 July 1986/Accepted 30 October 1986

Methanogens catalyze the hydrogen-dependent eight-electron reduction of carbon dioxide to methane. Twoof the key catalysts in the eight-electron reduction pathway are the nickel-containing enzymes F420-reducinghydrogenase and methyl reductase. In the present study, the structures of these archaebacterial enzymes fromMethanobacterium thermoautotrophicum AH have been determined by electron microstopy. By negative staintechniques, F420 hydrogenase was found to be a ring structure with a diameter of 15.7 nm and an inner channel4 nm in diameter. Shadow-casting experiments demonstrated that the rings were 8.5 nm deep, inditating aholoenzyme molecular weight of 8.0 x 105. Methyl reductase appeared to be an oligomeric complex ofdimensions 8.5 by 9 by 11 nm, with a central stain-penetrating region. The morphology and known subunitcomposition suggest a model in which the subunits are arranged as an eclipsed pair of open trimers. Methylreductase was also found in the form of larger aggregates and in paracrystalline arrays derived from highlyconcentrated solutions. The extremely large size of F420 hydrogenase and the methyl reductase supramolecularassemblies may have relevance in vivo in the construction of multiprotein arrays that function in methanebiogenesis.

Methanobacterium thermoautotrophicum AH is a thermo-philic member of the archaebacteria, a group thought to havediverged early in evolution from the more commonly studiedeubacteria (8). This bacterium meets all of its energy require-ments by oxidation of hydrogen gas (28). Furthermore, M.thermoautotrophicum utilizes carbon dioxide as both anelectron acceptor and a carbon source, producing methaneand less-reduced one-carbon fragments which get incorpo-rated into cellular macromolecules as follows:

90%4H2 + CO2 CH4T + 2H20

10%Cell Carbon

The unique CO2 reduction pathway is at the core of meth-anogenic biochemistry. It is possible that the methariogenicpathway has a long evolutionary history in this ancient groupof organisms. In this context, the intracellular structure andorganization of the proteins catalyzing these reactions are ofgreat interest.M. thermoautotrophicum AH possesses several unustal

cofactors and coenzymes (26), as well as four novel nickel-containing enzymes. Of these enzymes, two function ashydrogenases (12), and the third, methyl coenzyme Mreductase (2, 10), catalyzes the ultimate methane-yieldingreaction. The fourth nickel enzyme, CO dehydrogenase, iscentral in assimilation and dissimilation of acetate and twoone-carbon fragments at the methyl and carbon monoxideoxidation states (24). The two hydrogenases have beenshown to be distinct proteins by purification of each tohotnogeneity and by immunological methods (J. A. Fox,D. J. Livingston, W. H. Orme-Johnson, and C. T. Walsh,submitted for publication; L. Jordan, Ph.D. thesis, Massa-chusetts Institute of Technology, Cambridge, 1985). Onehydrogenase reduces the methanogen deazaflavin redox

* Corresponding author.

coenzyme, F420, and methyl viologen, whereas the otherhydrogenase reduces only methyl viologen. The propertiesof the bettet-characterized F420-reducing hydrogenase aresummarized in Table 1. Clearly, this protein plays a signifi-cant role in methanogenesis, as evidenced by its concentra-tion in the cell, its large size, and its efficiency as a hydrogenoxidation and F420 reduction catalyst and by inhibition ofmethanogenesis in crude extracts by anti-F420 hydrogenaseantibody (D. J. Livingston, L. P. Wackett, and C. T. Walsh,unpublished data). Another protein crucial to methanogene-sis is methyl coenzyme M reductase, which catalyzes thelast step in the CO2 reduction pathway as follows:

CH3S/-SO3 + 2e + 2H+ -* CH4O + HS,--SO3

The subunit structure (a202y2) of this major soluble protein(7) and the structure of its Ni tetrahydrocorphin cofactor,F430 (18, 19), have been elucidated (Table 1).

Despite the purification of these nickel-containing meth-anogen catalysts, much remains unknown with respect to invitro reconstitution of methanogenesis and the coupling ofCO2 reduction with energy-conserving reactions in vivo.These deficiencies are apparent with the observation thatcell disruption of M. thermoautotrophicum AH reducesmethanogenesis from the whole-cell rate of 7,000 nmol/m'inper mg of protein to the maximum observed rate of 20nmol/min per mg of protein in cell lysates (5). Possibleexplanations for this dramatic diminution in methanogenicrates, as discussed by Daniels et al. (5), involve destructionof a membrane or related vectorial processes, disruption ofrequisite in vitro protein-protein interactions, or dilution ofnecessary cofactors. The participation of internal tnem-branes in methanogenesis was invoked by Kell et al. (14) inthe methanochondrion hypothesis. However, Sprott et al.(21) demonstrated methanogenesis in the absence of internalmembranes, leaving the role of internal membrane-requiringprocesses questionable. The issue of potential protein-protein interactions between methanogenic enzymes has, todate, remained unexplored. In this context, the structure and

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ELECTRON MICROSCOPY OF METHANOGENIC ENZYMES 719

TABLE 1. Relevant information for F420 hydrogenase and methyl reductase from M. thermoautotrophicum AH

Intracellular % of Subunit SubunitPrshtcgopProtein localizati!on prtsoluble Holoenzyme mol wt mol wt, stoichiometry Prosthetic groups

F420Proteinlocalization protein14F420 hydrogenasea Soluble fraction 4 7.75 x 105 4.7 a8 I8 Y8 90 Fe

3.1 4.3 Ni2.6 5.6 Flavin adenine dinucleotide

Methyl reductase Soluble fractionb job 3.0 x 105b 6.8ad2 P2 y2b 1.7 F430d (Ni-tetrahydrocorphin)(Supramolecular form, 4.7

up to 9.6 x 106)c 3.8a Information on F420 hydrogenase from data of Fox et al., submitted.b Data from reference 7.c Data from this study.d Data from reference 11.

organization of key methanogenic enzymes has relevance inunderstanding in vivo function. F420 hydrogenase andmethyl reductase, two large multisubunit proteins that com-pose greater than 4 and 10%, respectively, of the totalsoluble cell protein are candidates for participation in anypotential supramolecular protein assemblies. The presentstudy was conducted in order to gain insight into thestructures of these enzymes. This was accomplished byelectron microscopy by using negative staining, image proc-essing, shadow casting, and immunoelectronmicroscopytechniques. In addition, supramolecular aggregates ofmethyl reductase in solution and in a paracrystalline arrayhave been examined in order to gain insight into possible invivo protein organization for these crucial methanogen en-zymes.

MATERIALS AND METHODS

Protein purification and characterization. The AH strain ofM. thermoautotrophicum (ATCC 29096) was cultivated asdocumented earlier (11). Enzymes were obtained from thisorganism as previously described; methyl reductase wasobtained by the method of Hausinger et al. (11), and F420-reducing hydrogenase was purified by using DEAE-Sephadex, Sepharose 6B, and mono Q fast protein liquidchromatography (Pharmacia, Inc., Piscataway, N.J.). Thedetails of the purification and the properties of the F420hydrogenase will be published elsewhere (Fox et al., submit-ted). Protein purity was assessed by 8% (methyl reductase)and 10% (hydrogenase) sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) by the procedure ofAmes (1).

Differential centrifugation of crude lysates. M. thermoauto-trophicum AH cells in suspension (1 g of cells per 1 ml of 50mM potassium phosphate [pH 7.0]) were disrupted byanaerobic passage through a French pressure cell. The crudelysate was centrifuged at 35,000 x g for 40 min at 4°C in aBeckman L3-40 ultracentrifuge. A portion of the supernatantfluid was diluted 1:3 (vol/vol) with 10 mM glycine-NaOHbuffer (pH 8.7) containing 10 mM MgCl2. Without anyattempt to exclude oxygen, the solution was further proc-essed by centrifugation at 160,000 x g for 2 h at 10°C in aBeckman L2-50 ultracentrifuge. The resultant pellet wassuspended in 1/10 volume of glycine-MgCl2 buffer and cen-trifuged in an Eppendorf microfuge for 2 min to sedimentlarge aggregated material.

Electron microscopy. Concentrated enzyme solutions (>1mg/ml) were diluted to 5 to 50 ,ug/ml with 10 mM Trishydrochloride (pH 7.0) before observation. A 10-,ul amountof the diluted protein solution was mixed with 10 [l of asuspension of bacteriophage T4 (106 phage per ml), which

was included as an internal standard for calibrating measure-ments of protein dimensions. One drop of the mixture wasplaced onto a glow-discharged, carbon-coated, 400-mesh,copper-coated grid for 15 s or 1 min. The sample was washedoff with 1 drop of water and immediately stained with 2%uranyl acetate or 2% phosphotungstic acid (pH 7.0) for 15 s.Most of the stain was removed with 1 drop of water, and thegrid was dried with the edge of a filter paper. Pictures weretaken with a JEOL 100B electron microscope operating at anaccelerating potential of 80 kV.

Undirectional shadow casting. F420 hydrogenase was ap-plied to grid surfaces as described above. The grids wereplaced in a Kinney evaporator, and a vacuum of less than10-6 torr was applied (1 torr = 133.322 Pa). The protein wasshadowed at an incident angle of 11.5 degrees by vaporiza-tion of a 2-inch length of a 0.008-inch-diameter platinum-palladium (80:20, wt/wt) wire (1 inch = 2.54 cm).Immunoelectron microscopy. Immunoglobulin G (IgG) ob-

tained from antisera raised against the a subunit of F420

AB..NW C

FIG. 1. SDS-PAGE analysis of proteins studied by electronmicroscopy. Shown are purified F420-reducing hydrogenase at 8 (A)and 15 p.g (B) and purified methyl reductase at 20 jig (C). df,Tracking dye front.

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720 WACKETT ET AL.

U 20 8

a-

cr 10

0 40 80 120 160 200

DIAMETER (AiFIG. 2. Electron micrographs of F420 hydrogenase. Particles

were negatively stained with uranyl acetate (magnification,x290,000) (A) or phosphotungstic acid (magnification, x270,000)(B). (C) Histogram of the frequency of measured diameters ofhydrogenase particles that had been negatively stained with uranylacetate.

FIG. 3. Electron micrographs of F420 hydrogenase and bacter-iophage T4 that were unidirectionally shadowed with Pt-Pd on thesame specimen grid: Shown at the top of the figure are selectedimages of shadows cast by F420 hydrogenase molecules, highlightedby circles and outlined in the upper left; shown at' the bottom of thefigure is a shadow cast by bacteriophage T4 that served as aninternal standard for making measurements. Magnification,x240,000.

hydrogenase was prepared as described by J. A. Fox et al.(submitted). F420 hydrogenase (440 ,ug; 1.1 ,uM) was incu-bated with anti-a subunit IgG (450 ,ug; 5.5 ,uM) in 10 mm Trishydrochloride (pH 7.0). After 1 h at room temperature, themixture was applied to a Sepharose 4B column (1 by 18 cm)and eluted with Tris hydrochloride (pH 7.0) at a rate of 20ml/h. The first fraction showing absorbance at 280 nm,enriched in cross-linked hydrogenase molecules, was exam-ined by negative-stain electron microscopy as describedabove.Data analysis. The diameter and inner channel of the F420

hydrogenase were measured from electron micrographs withfinal magnifications that ranged from x250,000 to x350,000.The surface area of the most commonly observed projection,as seen with negative staining, was deduced as follows. Printimages of the hydrogenase were traced out on the screen ofa Joyce-Loebl Magiscan 2. The surface area and statisticalanalysis of the data were both derived by using this system.The height of the hydrogenase was determined from directcomparison of observed shadows with the shadow cast by abacteriophage T4 tail (diameter of 16.6 nm) in the samephotographic field.The model for the methyl reductase structure was deduced

from photographs at x300,000 to x450,000 final magnifica-tion by aligning common structures and making clay modelsto match the observed projections.

J. BACTERIOL.

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.Ic

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10,I

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ELECTRON MICROSCOPY OF METHANOGENIC ENZYMES 721

FIG. 4. Composite immunoelectron micrographs of F420 hydrogenase stained with uranyl acetate after preincubation with anti-a subunitIgG (A) or preimmune IgG (B). Magnification, x 180,000.

RESULTS

Structural distinction between hydrogenases. M. thermo-autotrophicum AH contains two soluble hydrogenase activ-ities that separate upon fractionation of crude extract proteinby gel filtration on Sepharose 6B (12). Biochemical andimmunological evidence indicates that they are unique pro-teins. This contention was further supported by electronmicroscopy by using negative stain techniques. Partiallypurified preparations of the methyl viologen-reducing hy-drogenase showed most structures with dimensions (6 to 8nm) consistent with the calculated holoenzyme molecularweight of 2 x 105 (Jordan, Ph.D. thesis). No large structures(>10 nm) were observed. In contrast, the F420 hydrogenaseis known to be extremely large, with a molecular weight of7.75 x 105, as determined by gel filtration and native gelelectrophoresis. The F420 hydrogenase is available in higherpurity, is better characterized, and constitutes a more sig-nificant fraction of the total soluble protein than the methylviologen-reducing hydrogenase (see Table 1 for relevantproperties of the F420 hydrogenase). The structure of thisprotein was examined in some detail.F420 Hydrogenase. The two samples of F420 hydrogenase

used in this study were estimated to be >80% and >90%pure (Fig. 1A and B), as the latter sample contained onlysubunits attributed to the hydrogenase by SDS-PAGE when15 ,ug of protein was loaded.Negative staining with 2% phosphotungstic acid or 2%

uranyl acetate revealed similar structures (Fig. 2A and B).Uranyl acetate stained more uniformly and was used forfurther experiments. Fixation of the hydrogenase by prein-

cubation in buffer containing glutaraldehyde did not appearto increase structural uniformity.The predominant appearance of these structures is that of

an electron-transparent protein ring. It is not clear whetherthe electron-scattering center is due to a channel through thecenter of the protein or whether it represents an invaginationlarge enough to accumulate uranyl acetate. The predomi-nance of these apparent ringlike structures could be due to acommon appearance in several projections or to preferentialadherence of a particular surface(s) to the carbon-coatedgrid. Over 50 protein structures were measured in order to

A B

I

85 A

157 A

FIG. 5. Proposed model of F420 hydrogenase structure based ondata obtained by electron microscopy and from SDS-PAGE andnative gel electrophoresis. (A) The basic building block, an ac4ytrimer. (B) The 8.0 x 105-molecular-weight protein, composed ofeight otl3y trimers arranged as two stacked rings.

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722 WACKETT ET AL.

A B C D E_ ; .... I . t .. ;. ........... ! E.< ... m ...... ... ..l a .B : .: 4 ., s. . ... . .

C-

:---

f'i .1: iz!*.::..... I.

FIG. 6. SDS-PAGE analysis of the 35,000 x g supernatantfraction derived from a crude cell lysate (A), the 160,000 x g pelletderived from 35,000 x g supernatant fluid (B), the 160,000 x g

supematant fluid (C), purified F420 hydrogenase (D), and purifiedmethyl reductase (E). Arrows on the left point to the three subunitsof F420 hydrogenase. Arrows on the right show the positions of thesubunits of methyl reductase.

determine the size distribution of the observed ring struc-tures. Measurements were calibrated by the inclusion of T4bacteriophage as an internal standard in all photographicfields. The ring diameters, measured randomly with respectto orientation, were predominantly in the range of 14 to 17nm, with an average diameter of 15.7 ± 1.7 nm and an

electron-scattering channel of approximately 4 nm (Fig.2C).However, due to the irregularities in the shape of the

perimeter and the channel of the F420 hydrogenase rings, an

accurate surface area could not be deduced from the mea-surements noted above. In order to make this determination,photographic image processing was performed as describedin Materials and Methods. The ring surface area, determinedby this procedure, was 1.1 x 103 ± 2 x 101 nm2.These results suggested that the hydrogenase rested on the

grid surface in a preferred orientation or perhaps in twoorientations marked by a twofold symmetry axis. Images 8to 10 nm thick that could be interpreted as side views were

observed but were rare. To further probe the three-dimen-sional structure of the hydrogenase, the preparation was

examined by shadow casting. A value for the depth of thestructures shown in Fig. 2 would allow an estimation of theprotein volume and, hence, the protein molecular weight.The hydrogenase was unidirectionally shadowed on grids

containing bacteriophage T4 as an internal standard. As a

control for the proper assignment of a shadow to thehydrogenase ring, some grids were negatively stained withuranyl acetate before shadowing. The shadows from stainedhydrogenase molecules were more heterogeneous in lengththan were their unstained counterparts and were not used insize determinations. However, the shape of the shadow and

the density of images per grid were similar to the unstainedimages, thereby increasing our confidence that we werefocusing on the correct shadows. A characteristic triangularshadow with a relatively light buildup of metal at the base ofthe protein was observed (Fig. 3). Direct comparison of theshadows cast by the T4 tail with those cast by the hydroge-nase gave an average height of 8.5 + 1.5 nm for the proteinstructure. This result is indicative of a depth of approxi-mately one-half the width of the protein and is consistentwith a side projection being two subunits thick. Assuming,for calculation purposes, a uniform cylindrical projection of8.5 nm from the ring surface to the grid, the total volume ofthe protein is 9.47 x 102 ± 2.7 x 101 nm3. By using anaverage protein density of 1.4 g/cm3 (4, 16), this translates toa calculated molecular weight of 8.0 x 105 ± 2.3 x 104. Thisis similar to the value of 7.75 x 105 that had been determinedpreviously in our laboratories by electrophoresis and gelfiltration experiments (Fox et al., submitted).As a further confirmation that the ring structures were the

F420 hydrogenase, subunit-specific IgG was used to formaggregates that could be observed by electron microscopy.Anti-holoenzyme IgG had previously been shown to quanti-tatively precipitate hydrogenase activity (Fox et al., submit-ted), but the resultant heavy immunocomplex would beunsuitable for observation by electron microscopy. In thesame study, anti-a subunit IgG had been demonstrated tocross-react with the a subunit but not to immunoprecipitateholoenzyme. Thus, anti-a subunit IgG was selected as abetter candidate for cross-linking hydrogenase structuresinto small clusters. Preincubation of anti-a subunit IgG withF420 hydrogenase did indeed produce a collection of dimers,trimers, tetramers, and larger aggregates which had not beenobserved previously in the absence of antibody (Fig. 4A). Acontrol incubation conducted in parallel with preimmuneIgG failed to yield observable aggregates (Fig. 4B).A model of F420 hydrogenase consistent with the overall

dimensions of the F420 hydrogenase and the available infor-mation on subunit molecular weights and stoichiometry isshown in Fig. 5. Previously, activity staining of hydrogenasesubjected to native gel electrophoresis indicated that theminimal catalytic unit is a multisubunit complex with amolecular weight of 1.15 x 105. SDS-PAGE demonstratedthree classes of subunits with molecular weights of 4.7 x 104,3.1 x 104, and 2.6 x 104 (Fig. 1A and B); thus, the minimalunit is likely to be a trimer of subunits with a stoichiometryof 1:1:1 (1.04 x 105 molecular weight; Table 1). An octamerof trimers would assemble to form an aggregate of 8.3 x 105which is similar to the molecular weight of 8.0 x 105 deducedfrom electron microscopy size determinations. Thus, ourcurrent perception of the structure of F420 hydrogenase isthat of two stacked rings, each composed of a tetramer oftrimers (Fig. 5). The ridge depicted as spanning the regionbetween the channel and the outer perimeter was deducedfrom the observed pattern of metal buildup at the base of theprotein in shadowing experiments.

High-speed-sedimentable fractions. In a study by Doddemaet al. (6), 18% of the total methyl viologen-reducing hydrog-enase activity in M. thermoautotrophicum AH was found tosediment after centrifugation at 150,000 x g for 3 h. Thisresult, coupled with cytochemical staining of membranevesicles, was interpreted as supporting an association ofhydrogenase with a population of small membrane vesicles.At the time of this study, it was not yet known that twodistinct hydrogenases exist in M. thermoautotrophicum AH.It should be noted that both hydrogenases reduce methylviologen, whereas only one reduces F420 (12).

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ELECTRON MICROSCOPY OF METHANOGENIC ENZYMES 723

FIG; 7. Electron micrographs of the 35,000 x g supernatant fraction derived from a crude cell lysate (A) and the 160,000 x g pellet derivedfrom 35,000 x g supematant fluid (B). Circles denote hydrogenase molecules. Magnification for both panels is x 190,000, as determined fromincluded bacteriophage T4 tail sections (100 nm in length).

In light of these more recent findings, we conductedexperiments similar to those of Doddema et al. (6). Weobserved that high-speed centrifugation enriched the P and ysubunits of the F420-reducing hydrogenase with respect toother proteins, as determined by SDS-PAGE (Fig. 6). Itshould be noted that the a subunit of the hydrogenase is notwell resolved from the p subunit of methyl reductase; thus,it cannot be followed reliably by centrifugal fractionationand electrophoresis. In parallel with SDS-PAGE results, theconcentration of ring structures (15.7 nm in diameter) perunit protein increased after centrifugation at 160,000 x g(Fig. 7A and B). This experiment supports our identificationof the ring structure as the F420 hydrogenase and increasesour confidence that we have observed the native structure,as this material had been processed by centrifugation only.

Inspection of a large number of electron micrographs ofF420 hydrogenase which had been sedimented from 35,000x g supernatant fractions at 160,000 x g showed most of theprotein to be apparently freely dispersed, although someprotein appeared to be juxtaposed to membrane vesicles and

perhaps associated with other proteins. With the presentknowledge of the protein molecular weight (8.0 x 105), andassuming both a spherical structure for hydrodynamic cal-culations (4) and negligible interaction between protein mol-ecules, we can predict that 60% of the protein shouldsediment after being subjected to a centrifugal force of160,000 x g for 2 h. Thus, F420 hydrogenase would sedinmentindependently under the experimental conditions used, andthe question of its possible associations with other intracel-lular structures, although intriguing, remains unansweredpending further experimentationi.Methyl reductase in solution. Methyl reductase was puri-

fied anaerobically and displayed only the three bands attrib-uted to the protein after analysis by SDS-PAGE (Fig. 1C).Furthermore, the ratio of A278 to A418 was 8.9 to 1.0. Thesecharacteristics are similar to those of previous preparationsobtained in this laboratory and in others (2, 7, 11, 17).Methyl reductase was examined by electron microscopy

by using uranyl acetate as negative stain (Fig. 8). Sizedeterminations of negatively stained particles showed a class

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724 WACKETT ET AL.

A)BackTop

110 A

Side

90A

1 10 A

85 A

Frontiio A

T85 AB )

Side

FIG. 8. Electron micrographs of methyl reductase negativelystained with uranyl acetate (magnification, x280,000). This micro-graph shows structures determined to be the 3 x 105-molecular-weight form of methyl reductase, as well as particles twice that size.Some representative images of the 3 x 105-niolecular-weight formare highlighted with circles.

of structures that had dimensions consistent with the re-ported molecular weight of 3 x 105 (7). Some of thesestructures are highlighted with circles in Fig. 8. In addition,a class of molecules of approximately 6 x 105 molecularweight was also observed. An examination of over 100representative smaller images suggested two possible mod-els for the three-dimensional subunit structure of the a232 y2

protein (Fig. 9A and B). These models depict an unusualtrimeric structure which is open, rather than being organizedin a closed ring structure. The observed symmetry of thetop, front, and back projections is consistent with the o43y

trimers being in an eclipsed, rather than staggered, configu-ration with respect to each other. The resolution of theseobservations was not sufficient to discriminate between acommon subunit hinge with an opening at the ends of thetrimer "V" (Fig. 9B) and one with an opening at the vertexof the "V" (Fig. 9A). Although these models show a distinctarrangement of subunit pairs, we cannot assign the at,,, or y

subunits to their respective positions on the basis of currentinformation.Methyl reductase high-molecular-weight aggregates. In ad-

dition to the previously described 3 x 105-molecular-weightunit observed by negative staining, considerably largerstructures were observed in some electron micrographs (Fig.10). These highly symmetrical particles were sometimesobserved upon diluting a highly concentrated solution ofhomogeneous methyl reductase (100 mng/ml in 10 mM potas-sium phosphate buffer [pH 7.0]) into 10 mM Tris buffer just

{85A

90A

Front

110 A

}85 A

FIG. 9. Two proposed models (A and B) of methyl reductasesubunit arrangement based on observation of negatively stainedpreparations. For the model in panel A, four projections are shown.For the model in panel B, two projections are shown in order toillustrate that the models in panels A and B differ only by theposition of the groove observed between subunits. It should benoted that the assignment of a specific subunit to any of the threepossible positions is not implied in this model.

before application to a grid. Although definitive identifica-tion of these larger structures as methyl reductase has yet tobe done, an apparent gradient of structures that could becomposed of the 3 x 105-molecular weight unit is visible inFig. 10. Nearly spherical aggregates with diameters as greatas 28 nm have been observed. If these are indeed solidprotein spheres, they represent methyl reductase aggregatesof molecular weights approaching 9.6 x 106.

Crystalline methyl reductase. During the course of concen-trating a solution of methyl reductase, yellow crystals wereobserved at the periphery of the concentrate. A crystal wasdissolved in Tris buffer, and SDS-PAGE analysis of thissolution showed the characteristic a, I, and y subunit bandsof methyl reductase. An examination of this solution byelectron microscopy revealed structures resembling the 3 x105-molecular-weight unit of methyl reductase, as well asmany larger particles reminiscent of the aggregates de-scribed above.We attempted to determine the structure of the protein

molecules within these paracrystalline aggregates. The macro-scopic crystals were many layers thick and so necessitatedmechanical disruption before direct examination by electron

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ELECTRON MICROSCOPY OF METHANOGENIC ENZYMES 725

FIG. 10. Electron micrograph of methyl reductase, showing supramolecular aggregates. Nearly spherical structures of diameters up to 20nm are highlighted with circles. The preparation was negatively stained with uranyl acetate. Magnification, as deduced from includedbacteriophage T4 tail sections (100 nm in length), was x300,000.

microscopy. A crystal was disrupted on a glass slide with a25-gauge syringe needle, blotted with a carbon-coated grid topick up some fragments, and stained with uranyl acetate.The smooth but layered structure of this protein assemblywas readily apparent by examining the surface and edges ofa fragment at low magnification (Fig. 11A). Upon closerinspection (Fig. llB), molecular detail was revealed, and athigh magnification (Fig. liC), the full nature of the compo-nent structure was apparent. The building blocks of theparacrystalline aggregate resembled some of the structuresobserved with mnethyl reductase in solution (Fig. 10). Thissecond observation of methyl reductase in a large assemblyis suggestive of subunit binding sites that allow self-aggregation.

DISCUSSION

In the complex hydrogen oxidation and carbon dioxidereduction chemistry they carry out, methanogens utilize twonickel-containing hydrogenases and methyl reductase,which contains the nickel tetrahydrocorphin cofactor F430.The importance of the F420-reducihg hydrogenase andmethyl reductase in M. thermoautotrophicum AH is under-scored by their presence in amounts of 4 and 10%, respec-tively, of the soluble protein. Furthermore, their distributionand general size characteristics may be conserved within themethanogen class (13, 27; P. Hartzell and R. S. Wolfe,Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, 112, p. 141).

Previous studies had established that F420-hydrogenase iscomposed of three distinct subunits that may be arranged ina 1:1:1 stoichiometry, as suggested by the observation of aminimal catalytic unit with a molecular weight of 115,000 by

native PAGE followed by activity staining (Fox et al.,submitted). From this result and from the structural detailselucidated in the present study, the holoenzyme of 8.0 x 105molecular weight might be composed of an a8P8Ps8 subunitstructure. Given the complexity of this structure, possiblycomposed of 24 subunits, further studies are required toestablish a detailed subunit map. Subunit-specific antibodieswould be helpful in this regard, as their utility is documentedin ribosome structure elucidation (15) and, more recently,with photosynthetic coupling factor 1 (25).Two models of the methyl reductase structure that are

consistent with the electron microscopy data depict two ca4yunits attached at one or two subunit hinges (Fig. 9A and B).The assignment of a particular subunit to a given positioncannot be made on the basis of the present data, butsubunit-specific antibodies could also prove useful in thisregard.

Starting with the postulated methyl reductase structure(t2P2Y2), one can construct aggregates (e.g., a molecularweight of 9.6 x 106 = 32 (X2P2Y2 units) that would appear tobe nearly spherical by the negative staining technique. Itshould be noted that riboflavin synthase is a catalytic proteinthat forms a capsidlike arrangement of 60 p subunits inapparent icosahedral symmetry (3). Filament formation byglutamine synthase (23) and the paracrystalline aggregationof ribulose-bisphosphate carboxylase in carboxysomes (20)are further examples of elaborate protein aggregates. An-other biological comparison, perhaps germane to in vivostructure and function relationships of methanogen proteins,may be the protein milieu of the mitochondrial matrix. In thisenvironment, in which protein concentrations may reach upto 400 mg/ml (22), substrate channelling and paracrystalline

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726 WACKETT ET AL.

FIG. 11. Electron micrograph of a mnechanically disrupted crystal of methyl reductase. Shown are micrographs of the crystal stained withuranyl acetate and magnified x47,000 (A), x105,000 (B), and x200,000 (C).

multienzyme arrangements have been observed. It was at a

protein concentration resembling that of the mitochondrialmatrix that methyl reductase "paracrystallization" was ob-served.The large size of F420 hydrogenase and the methyl

teductase aggregates may have potential in vivo significancein the construction of a multicomponent complex that func-tions in methane biogenesis. A number of disparate obser-

vations support the contention that protein-protein interac-tions are important in methanogenesis. The reproducibilityof chromatographic separations of methanogenic proteins is

- strongly affected by column dimensions and flow rates,indicative of protein-protein interactions competing withprotein-matrix ihteractions (unpublished observations).Rates of in vitro methanogenesis do not vary linearly withprotein concentration; this suggests that dilution may cause

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ELECTRON MICROSCOPY OF METHANOGENIC ENZYMES 727

dissociation of protein aggregates to limit reaction rates.Furthermore, the RPG effect (9), in which the methylreductase reaction enhances rates of earlier steps inmethanogenesis, may be mediated through protein-proteininteractions. In this context, the structures of importantmethanogenic enzymes and their interactions and localiza-tion in vivo may well unlock the door to the major questionthat confounds current efforts to study methanogenesis invitro: why does cell lysis diminish methanogenic activity by99.7%?

ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grantGM 31574 from the National Institutes of Health (NIH) and an NIHpostdoctoral fellowship (GM 10342) to L.P.W.We thank Neil Bastian and Judy Fox for providing samples of

purified F420 hydrogenase and David Livingston for providinganti-a subunit F420 hydrogenase IgG. We acknowledge Rolf Thauerfor helpful discussion and Gregory Petsko for examining methylreductase crystals by X-ray diffraction techniques and suggestingfurther examination by electron microscopy.

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