HYDROGENASE AND HYDROGEN METABOLISM IN MICROCOCCUSAEROGENES1

WALDO CURTIS2 AND E. J. ORDALDepartment of Microbiology, University of Washington, Seattle, Washington

Received for publication March 11, 1954

The capacity of utilizing or of producingmolecular hydrogen is widespread among micro-organisms. An indication that a specific enzymesystem might be concerned in the metabolism ofhydrogen came from the demonstration by Taussand Donath (1930) that a bacterial culture grownautotrophically on H2, 02, and C02 reducedmethylene blue in the presence of molecularhydrogen. Subsequently, Stephenson and Stick-land (1931) showed that a number of hetero-trophic bacteria, mainly from the enteric group,were able to catalyze the reduction of methyleneblue, fumarate, nitrate, and oxygen by means ofmolecular hydrogen. The activation of molecularhydrogen was considered to be mediated by anenzyme which was named "hydrogenase."Hydrogenase, as measured by catalytic hydroge-nations of such substances as methylene blue,nitrate, sulfate, carbonate, oxygen, amino acids,and dicarboxylic acids, has been demonstrated innumerous microorganisms (Farkas and Fischer,1947). However, the reduction of methylene blueby molecular hydrogen has been employed mostfrequently as a test for hydrogenase.A number of bacteria containing hydrogenase,

as measured by hydrogenation procedures, havebeen found to catalyze the exchange of deuteriumwith water, i.e., D2 + H20 = HD + HDO(Farkas, 1936; Hoberman and Rittenberg, 1943;Farkas and Fischer, 1947; Johnston and Frenkel,1951; Hyndman et al., 1953). In addition, fixa-tion of molecular tritium by a variety of bacteriahas been demonstrated by Smith and Marshall(1952).

It is logical to consider that catalysis of theexchange reaction is a fundamental property ofhydrogenase, and that the exchange reaction is a

1 Supported in part by the Atomic EnergyCommission and by the State of Washingtonfund for research in biology and medicine.

2 U. S. Public Health Service PostdoctorateResearch Fellow of the National MicrobiologicalInstitute.

true measure of hydrogenase activity. However,most experiments on hydrogenase have been car-ried out using hydrogen acceptors, and as notedby Hyndman et al. (1953) it is quite possible thatthe interpretation of results is complicated bythe necessity of intermediate electron carriers orof enzymes for activation of the acceptors. Whilethis complication is obvious with acceptors suchas nitrate, fumarate, and oxygen, it is not certainthat it exists with an acceptor such as methyleneblue.Molecular hydrogen is a metabolic end product

of a large number of microorganisms of widelydifferent physiological types. A number of theseproduce molecular hydrogen from formate asfirst shown by Pakes and Jollyman (1901) in thecase of Bacillus coli (Escherichia coli). How-ever, there is disagreement as to whether hydroge-nase is involved in the production of molecularhydrogen. Stephenson and Stickland (1932) de-scribed an enzyme, formic hydrogenlyase, whichwas considered a specific enzyme not related tohydrogenase, and which was considered solelyresponsible for the evolution of molecular hydro-gen from formic acid. This conclusion was basedon the finding that four strains of Bacillus lkcti8aerogenes (Aerobcter aerogenes) were negative forhydrogenase, as measured by the reduction ofmethylene blue, yet were able to produce molecu-lar hydrogen from formate. Similar findings havebeen reported recently by Grunberg-Managoet al. (1951) and by Lichstein and Boyd (1953).On the other hand, Ordal and Halvorson (1939),Ordal and Tsuchiya (1940), Hoberman andRittenberg (1943), Waring and Werkman (1944),DeLey (1951), and Gest (1952) have providedevidence that the enzyme hydrogenase is anecessary component of the enzyme systemresponsible for the evolution of molecularhydrogen from formate.

Although hydrogenase has been found in themajority of the microorganisms liberatingmolecular hydrogen, there is little evidence on the

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question of involvement of hydrogenase in theevolution of molecular hydrogen from substratesby bacteria which are unable to decomposeformic acid. Koepsell and Johnson (1942) instudying the decomposition of pyruvate toacetate, carbon dioxide, and hydrogen by cell-free preparations of Clostridium butylicumreported that hydrogenase was absent, asmeasured by reduction of methylene blue. On theother hand, the enzyme was present in intactcells of C. butylicum.Miococus aerogenes (Whiteley, 1951) fer-

ments pyruvate with production of molecularhydrogen and other products but is unable todecompose formate and is devoid of hydrogenaseas judged by catalytic hydrogenation of suchsubstances as methylene blue, fumarate, fern-cyanide, nitrate, amino acids, and oxygen. Thefailure to demonstrate hydrogenase in M.aerogenes by the conventional hydrogenationtechnique would imply that hydrogenase doesnot participate in the evolution of hydrogenfrom pyruvate by this organism. However, thereremains the possibility that hydrogenationtechniques do not necessarily give a true measureof the activity of the enzyme hydrogenase. Thequestions of the presence of hydrogenase inM. aerogenes and its involvement in the evolutionof molecular hydrogen from pyruvate are ex-plored in the present investigation.

EXPERIMENTAL METHODS

Micrococcue aerogenes, strain 228, was obtainedfrom the lyophilized culture collection at theUniversity of Washington and cultivated intubes of the following medium: tryptone (Difco),1.0 per cent; yeast extract (Difco), 1.0 per cent;sodium glutamate, 0.4 per cent; K,HPO4-3H1O,0.3 per cent; sodium thioglycolate, 0.1 per cent;agar, 0.05 per cent; methylene blue, 0.0002 percent; and tap water. In order to obtain largeamounts of cells, two liter quantities of the samemedium, but lacking the methylene blue andagar, were inoculated with 50 ml of a 12 to 20hour culture. The cells were harvested after 16to 20 hours of incubation at 37 C and washed bycentrifugation from m/15 phosphate buffer at pH7.0, containing 0.01 per cent sodium sulfide or0.02 per cent cysteine. Extracts of cells wereprepared by grinding for five minutes withalumina (Mclwain, 1948). The alumin-ellpaste then was extracted with 0.1 per cent

Figure 1A-water decomposition systemB-manifold for introducing deuteriuma-water decomposition vesselb-trapc, c-gas sampling vesselsd, d-manometerse, e-ball and socket jointsf-reaction vesselg-to deuterium storage tankh-to trap and pumpOperation. The reaction vessels (f) containing

cell extract or suspension, buffer, etc. wereconnected as shown in the figure and evacuatedand filled with deuterium, then re-evacuated andfilled a second time to about 740 mm mercury.The joints at (e) were disconnected then and thereactions vessels and connections put on a shakerin a water bath at 30 C. To withdraw a samplefrom a reaction vessel (f), the vessel was removedfrom the bath and inverted to fit on joint (i) ofthe water decomposition vessel containing aboutone gram metallic sodium. System A was evacu-ated then to joint (j) and the stopcock (k) closed.Stopcock (j) then was opened to admit about 1.5ml liquid to the tube above stopcock (k). Stop-cock (j) was closed then and the seal at joint (i)broken, the residual liquid above (j) removed,and the reaction vessel was returned to the shaker.The liquid above (k) was agitated then with airfrom a syringe to remove dissolved deuteriumand slowly admitted to vessel (a) where it reactedwith sodium to give gaseous hydrogen whichthen passed through trap (b) and was collected invessel (c). The contents of vessels (c) wereanalyzed in the mass spectrometer. In someexperiments individual reaction vessels (f) wereused for each reading.

aqueous solution of sodium sulfide or 0.02 percent cysteine adjusted to pH 7.0, using approxi-mately 5 ml solvent per gram of cells, andcentrifuged at 10,000 rpm to remove aluminaand cellular debris. The protein content was

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estimated by the trichloracetic acid method ofStadtman et al. (1951).

Tests for hydrogenase activity were carriedout both by hydrogenation and by deuteriumexchange. The hydrogenation experiments werecarried out in a conventional Warburg apparatusunder an atmosphere of hydrogen. Each reactionvessel ordinarily received 0.6 ml of a 10 per centsuspension of cells or 0.6 ml of cell-free extractand 1.0 ml of m/10 phosphate buffer at pH 6.2.From 3 to 10 micromoles of various substrateswere tested as potential hydrogen acceptors.Assay of hydrogenase also was attempted byreduction of methylene blue using the Thunbergmethod.The apparatus diagrmed in figure 1 was

employed in the exchange studies. The gas phaseinitially was 99+ per cent deuterium, and theliquid phase normal water. Samples of the liquidphase were taken at intervals and converted togaseous hydrogen by the action of metallicsodium. Hydrogen-hydrogen deuteride ratios ofthe resulting gas were measured then on aConsolidated-Nier isotope-ratio mass spec-trometer. In some early experiments the gasphase was analyzed rather than the liquid phase.

RESBULTS

M. aerogenes, strain 228, ferments pyruvicacid, a number of amino acids, purines, andpyrimidines (Whiteley, 1951, 1952) with forma-tion of varying amounts of molecular hydrogen,carbon dioxide, lactic acid, and volatile acids. Inaddition ammonia is produced from the nitrog-enous compounds. Sugars and formate are notfermented. We have confirmed the finding byWhiteley that carbon dioxide and hydrogen arenot produced from formate by cells and extractsof M. aerogenes, and that hydrogen and carbondioxide are produced rapidly from pyruvate.

Thunberg tests for dehydrogenases. Tests for thepresence of hydrogenase by means of the Thun-berg technique with methylene blue werenegative. Suspensions of several densities wereused in these tests. Formic dehydrogenase wasabsent. However, it was found that pyruvatereduced methylene blue readily in the Thunbergtest. Typical results of a Thunberg test are givenin table 1. The reducing agent was omitted fromthe phosphate buffer used for washing andsuspending of the cells.

Hydrogenase assay by hydrogenation. Attempts

TABLE 1Thunberg test for dehydrogenases in

Micrococcw aerogenes

THUNB:RG TUBEREAGENTS

1 2 3 4

1:5000 methylene 0.5 0.5 0.5 0.5blue, ml

m/10 phosphate 2.0 2.0 2.0 2.0buffer, pH 6.2,ml

M/20 sodium for- 0.5mate, ml

M/20 sodium py- 0.5ruvate, ml

H20, ml 0.5 0.52 per cent cell sus- 0.5 0.5 0.5 0.5

pension, mlAtmosphere H2 vacuum vacuum vacuumTime of decolori- 102 106 86 7%

zation, minutes

at demonstration of hydrogenase by directmeasurement of hydrogen uptake in Warburgmanometers using methylene blue as substratewere negative. Similar tests with ferricyanide,fumarate, nitrate, oxygen, and a number ofamino acids were also negative.The apparent absence of hydrogenase in M.

aerogenes is in sharp contrast to the situationwith the taxonomically related Micrococcuslactilyticus, which posses a powerful andrelatively stable hydrogenase as measured byhydrogenation of methylene blue (Witter, 1953).M. kactilyticwus ferments pyruvate to acetate,propionate, carbon dioxide, and molecularhydrogen, but like M. aerogenes is unable todecompose formate (Whiteley, 1951).The negative results of tests for hydrogenase

in M. aerogesw, as measured by conventionalhydrogenation techniques, could be interpretedas evidence to support the hypothesis thathydrogenase is not a functional component of theenzyme systems responsible for the evolution ofmolecular hydrogen from pyruvate and othersubstrates. However, this interpretation shouldnot be accepted without exploring fully othermethods of assay of hydrogenase.Hoberman and Rittenberg (1943) using cell

suspensions of Proteus vulgaris found that 2 percent urethane inhibited the reduction of methyl-ene blue with molecular hydrogen, though the

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c gas phase with H2 due to decomposition ofpyruvate would be expected to give fictitiouslyhigh values for exchange. However, on calculation

A it was found that the D2/H2 ratio of the gas< ~> phase would change from 0.1434 to 0.1420 if it

were assumed that H2 was evolved from pyruvatein stoichiometrical proportions. Since the increasein exchange observed was considerably greater,

\8 pyruvate may be considered to act as a sparker.In later experiments on exchange a different

procedure was used, as noted in the section onMethods. The gas phase was 99+ per cent

60 120 130 deuterium, and the liquid phase initially con-MINUTES tained normal water. Measurements were made

Figure B. Deuterium exchange in Micrococcusaerogenes.The change in the D2/H2 ratio of the gas phase:A. 15 ml 16 per cent cell suspension in phos-

phate buffer at pH 6.2B. 15 ml 16 per cent suspension plus 10 Lm

pyruvateC. Buffer control

exchange reaction with deuterium was unaffected.It was suggested that an enzyme system inaddition to hydrogenase was involved in thereduction of methylene blue by molecularhydrogen. Hence it seemed possible that thereduction of methylene blue, as well as otheracceptors, by molecular hydrogen might requirethe presence of enzymes or cofactors which are

absent in M. aerogenes but which are present inmicroorganisms which show a positive test forhydrogenase by hydrogenation techniques. Ifthis is an adequate explanation, the exchangereaction with deuterium might be expected totake place since this involves the biologicalactivation of the hydrogen molecule withoutaddition of external electron acceptors.Hydrogenase assay by deuterium exchange. The

results of an early experiment on deuteriumexchange using a washed cell suspension ofM. aerogenes are given in figure 2. The gasphase contained a mixture of deuterium andhydrogen in the ratio 0.1434 at an initial pressureof 150 mm mercury. Since the aqueous phase hadno excess deuterium, it was expected thatshould exchange occur, the gas phase would bediluted with HD and H2. That this was the casecan be seen in curve A in figure 2. Curve Billustrates the effect of the addition of 10 micro-moles of pyruvate to the system. An apparentincrease in exchange occurred. Dilution of the

on the liquid phase after conversion to gas withsodium. Under these conditions dilution of thegas phase by normal H2 from pyruvate did notinfluence the experimental results.Both cell suspensions and cell-free extracts of

M. aerogenes were found to carry out deuterium

250

225'

" 200

to 75

150

Z 125

100

75

A

60 120 180 240MINUTES

Figure 3. Deuterium exchange in Micrococcusaerogenes and inhibition by methylene blue.

A. 6.0 ml 10 per cent cell suspension, 5.0 mlM/15 phosphate, pH 6.2, 1.0 ml H20

B. 6.0 ml cell-free extract (6 mg protein/ml),5.0 ml M/15 phosphate, pH 6.2, 1.0 ml H20

C. As B except that 1.0 ml M/25 methylene blueadded initially to give a final concentrationof M/300

D. As B except that 1.0 ml m/25 methylene blueadded at 50 minutes

-k

W.

0

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exchange. This is illustrated in figure 3. Theexchange with the whole cells of M. aerogenes(curve A) was fully as great as that shown by asuspension of Escherichia coli of somewhatgreater density which was found to possess apowerful hydrogenase by hydrogenation ofmethylene blue. The exchange carried out by thecell-free extract of M. aerogenes was relativelylow, but definite, as indicated in curve B.

The effect of methylene blue on deuteriumexchange. Since attempts to demonstrate hy-drogenase by means of hydrogenation of methyl-ene blue were negative, it was of interest todetermine whether this dye exerted any effect onthe exchange reaction. Curves C and D in figure3 show respectively the effects of addition ofmethylene blue initially, and after 50 minutes tocell-free extracts. Addition of methylene blueled to the immediate cesation of exchangeactivity. The concentration of methylene blueemployed (M/300) is approximately the same asthat used for assay of hydrogenase in the War-burg technique.Methylene blue inhibited deuterium exchange

by intact cells as well as cell-free extracts ofM. aerogenes. The effect of addition of m/260methylene blue on the exchange activity ofintact cells' of M. aerogenes is shown in figure 4.Addition of methylene blue initially (curve A)or after 78 minutes (curve B) stopped exchangeactivity. In similar experiments it was foundthat exchange activity of whole cells was stoppedby methylene blue in final concentrations ofM/250 and M/400.

The stimulation of deuterium exchange bypyruvate and benzyl viologen. The addition ofpyruvate increased the exchange activity ofintact cells of M. aerogener. This was illustratedin figure 2. With active cell suspensions only amoderate increase in activity occurred (see curvesC and D, figure 4).The exchange activity in cell-free extracts was

as a rule low, as compared with the activityshown by intact cells. This appeared to be due,at least in part, to reversible inactivation of theenzyme during the course of preparation. Theaddition of pyruvate consistently and stronglyincreased the activity of cell-free extracts ofM. aerogenes. Also the exchange activity ofextracts was usually, though not always, in-creased by the addition of small amounts ofbenzyl viologen. It was noted in a few instances

60 120 180 240MINUTES

Figure 4. The inhibition of deuterium exchangeby methylene blue.

A. 10 per cent cell suspension plus M/260methylene blue

B. 10 per cent suspension plus delayed dump ofM/260 methylene blue

C. 10 per cent suspension onlyD. 10 per cent suspension plus M/200 pyruvate

that a slight increase in exchange activityoccurred in initially inactive extracts withoutaddition of pyruvate or benzyl viologen when theperiod of incubation with deuterium was aroundfour hours or longer.An experiment showing the effect of addition

of pyruvate or of benzyl viologen on the exchangeactivity of a cell-free extract of M. aerogenes oflow activity is illustrated in figure 5. Clearly theaddition of pyruvate (curve C) stimulatesexchange activity as compared with that shownby the extract alone (curve A). The stimulatoryeffect of benzyl viologen is much less (curve B).As noted above, the small increase in activity ofthe extract alone at 240 minutes may have beendue to reactivation by deuterium.

The hydrogenation of methyl violet and benzylviologen. As judged by the exchange reaction,hydrogenase was present in cells and extracts ofM. aerogenes. Yet no indication of the presence ofhydrogenase was found with methylene blue,either by the Thunberg technique or by measure-ment of hydrogen uptake in Warburg manom-eters, and methylene blue was found to inhibit

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, 2503' C.S6200

1 50

1~~~~~~~ B.~~~~~~~1

A50

60 120 IS0 240MINUTES

Figure 6. The stimulation of deuterium ex-change in a cell-free extract by pyruvate andbenzyl viologen.A. Cell-free extract (6 mg protein/ml)B. Cell-free extract plus m/1,000 benzyl

viologenC. Cell-free extract plus M/100 pyruvate

the exchange reaction. In view of these observa-tions and in view of the sparking effect of benzylviologen on the exchange activity of cell-freeextracts (see figure 5), it was decided to test forhydrogenase by hydrogenation techniques usingbenzyl viologen and other oxidation-reductionindicators with standard potentials near that ofthe hydrogen-hydrogen ion couple. Only a fewcompounds of this class were available, and anumber of other possible acceptors also weretried. The potential acceptors tried includedmethylene blue, ferricyanide, glycine, proline,hydroxyproline, tryptophan, riboflavin, cresylblue, neutral red, resazunn, janus green, acri-flavin, phenosafrinin, methyl violet, methyleneviolet, and benzyl viologen. The tests werecarried out by using the Warburg techniquemeasuring uptake of molecular hydrogen in thepresence of 3 to 10 micromoles of the varioussubstrates.

Definite hydrogen uptake was obtained onlywith benzyl viologen and, surprisingly, methylviolet (figure 6). The hydrogen uptake did notreach stoichiometrical proportions and variedsomewhat with different suspensions. A smallamount of hydrogen was sometimes produced inthe endogenous controls.

Cell suspensions of M. lactilyticue and E. coli,possessing hydrogenase as measured by hy-drogenation of methylene blue, also take upmolecular hydrogen with benzyl viologen andmethyl violet. However, with both these organ-

isms the hydrogen uptake with methyl violetoccurs at a slower rate than with methylene blue.The demonstration of hydrogenation of benzyl

viologen and methyl violet, even though there isno hydrogenation of methylene blue, ferricyanide,etc., coupled with deuterium exchange activityshows clearly that an enzyme possessing thefundamental properties of hydrogenase is presentin M. aerogenes.

The effect of concetration of methhyiee blue onthe hydrogenation of methyl violet and benaylviologen and on deuterium exchange. It was notedearlier that methylene blue in concentrations ofm/260 to m/400 inhibited the deuterium exchangereaction with cell uspensions of M. aerogenes.Hence it was of interest to determine whethermethylene blue had any effect on the hydrogena-tion of methyl violet and benzyl viologen. Severalconcentrations of methylene blue were tested fortheir effect on hydrogen uptake with benzylviologen and methyl violet in concentrations ofm/250 (8 micromoles per vessel). It was foundthat m/250 and m/500 methylene blue causedcomplete inhibition of hydrogen uptake withbenzyl viologen and with methyl violet. With

20 40 60 80 100 120MINUTES

Figure 6. The hydrogenation of methyl violetand benzyl viologen by cell suspensions of Micro-coccus aerogenes.

A. m/250 methyl violet (8 micromoles)B. M/500 methyl violet (4 micromoles)C. x/250 benzyl viologen (8 micromoles)D. m/5OO benzyl viologen (4 micromoles)E. Endogenous control

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lSO.

1~ 25

;d 100

0 20 40 60 80 900

MINUTES

Figure 7. Methylene blue inhibition of hy-

drogenation of methyl violet by Micrococcusaero.

A. x/250 methyl violet

B. M/250O methyl violet plus iu/4,OOO methylene

blue

C. x/250 methyl violet plus M/2,O0O methylene

blueD. Endogenous control

lower concentrations of methylene blue, hyirogenuptake ordinarily took place after lag periods

roughiy proportional to the concentrations of

nethylene blue employed, though there was a

good deal of variation between different cell

pensions. With a given suspension,tmhe lagperiods were quantitatively les with benzylviologen thanl with methyl violet. Figue 7

illustrates the effect of M/2,OOO and x/4,OOOmethylene blue on the hydrogen uptake with

methyl iolet. Clearly the lag period before

hydrogen uptake occurs is dependent upon the

concentration of methylene blue used, though the

subsequent rates of hydrogen uptake (curvres Band C) are not significantly different from that

of the control (curve A).

An experiment on the effect of low concentra-tions of methylene blue on deuterium exchange issown in figure 8, using an aliquot of the cell

suspension used in the pring experiment. Thecell suspesion used in the experiments illustrated

in figures 7 and 8 was washed two times withfreshiy boiled and cooled buffer, with no reducingagent added. As shown in curve A of figue 8,

the suspension compared favorably in exchangeactivity with suspensions used in other experi-ments which were washed and supended inbuffer conting cysteine or sodium sulfide. Thesuspension containing M/4,000 methylene blue(curve B, figure 8) showed a rate of exchangeequivalent to that of the control after a lagperiod. Only a trace of color was found onexamination after 34 minutes. The first aliquotwas taken for analysis at 36 minutes, and at thistime exchange activity had begun. The suspensioncontaining M/2,000 methylene blue (curve C,figure 8) showed no exchange activity at 33minutes. The methylene blue was found de-colorized on examination at 65 minutes, and thesuspension showed a definite exchange in the finaltwo determinations. No exchange occurred with,a boiled s n of cells or with a suspensionof cells with m/1,000 methylene blue.

It has been established that cell-free extractsand suspensions of M. aerogenee do not reducemethylene blue with molecular hydrogen. Hencethe reduction of dilute concentrations of methyl-ene blue must be due to reducing substances inthe cell suspensions. The experiments on theeffect of methylene blue on hydrogen uptakewith benzyl viologen and methyl violet showthat high concentrations of methylene blue

200

175

$450

125

*100

75 ,q

50~~~~~0 40 80 120 160 200

MINUTESFigure 8. The effect of concentration of methyl-

ene blue on inhibition of deuterium exchange byMicrococcus aerogenes.

A. Suspension onlyB. Suspension plus m/4,000 methylene blueC. Suspension plus m/2,000 methylene blueD. Suspension plus M/1,OOO methylene blue

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inhibit the hydrogenation of these substrates;though with low concentrations, hydrogen uptakeoccurs after a lag period. In the presence ofbenzyl viologen and methyl violet it is of coursenot determinable whether the methylene blue isreduced when the hydrogen uptake begins. Theexperiment on the effect of dilute concentrationsof methylene blue (figure 8) on deuteriumexchange suggests strongly that this reaction isinhibited by methylene blue in the oxidizedform but that once the methylene blue is reducedthe inhibition disappears. With dilute methyleneblue there is a considerable degree of corre-spondence between the lag periods which occurbefore exchange and the hydrogenation reactionsbegin, and it is logical to conclude that theinhibitory action of oxidized methylene blue isat the same site, i.e., the enzyme activatingmolecular hydrogen.

The qustion of involvement of hydrogenaose inevolution of hydrogen from pyruvate. If hy-drogenase is involved in the evolution of mo-lecular hydrogen from pyruvate, it would beexpected that this reaction would be blocked byconcentrations of methylene blue similar tothose which block the exchange reaction. Ofcourse, some other essential enzyme might beeven more sensitive to methylene blue.

Experimentally it was found that methyleneblue effectively blocked the production of molec-ular hydrogen from pyruvate. The inhibitionwas reversible since when pyruvate was presentin excess amounts, molecular hydrogen was pro-duced from pyruvate following reduction of themethylene blue to the leuco form.The results of a Warburg experiment on the

effect of methylene blue on evolution of hydrogenfrom pyruvate are given in figure 9. Curve Arepresents hydrogen evolution from 10 micro-moles of pyruvate. Curve B represents hydrogenproduction from 10 micromoles of pyruvate inthe presence of 8 micromoles of methylene blue(concentration M/250). The methylene blue wasadded to the cell suspension when the cups wereprepared. At 60 minutes the methylene blue wascompletely reduced and hydrogen evolution hadbegun. The small amount of hydrogen producedcorresponded roughly to the difference betweenthe amounts of pyruvate and methylene blueused in this experiment.Curve C is essentially a duplicate of curve B

except that at 60 minutes, following reduction of

0 20 40 60 80 100 120MINUTES

Figure 9. The effect of methylene blue onproduction of hydrogen from pyruvate.

A. 10 micromoles pyruvateB. 10 micromoles pyruvate plus 8 micromoles

methylene blueC. 10 micromoles pyruvate plus 8 micromoles

methylene blue plus an additional 10 micro-moles pyruvate at 60 minutes

D. Endogenous control

the methylene blue, a second aliquot of 10micromoles of pyruvate was added from a sidearm. The resulting rate of hydrogen productionwas rapid, corresponding closely to that shownby the control (curve A) containing pyruvatebut without methylene blue.

In another experiment it was found that carbondioxide was produced rapidly and without lagfrom pyruvate in the presence of methylene blue,and that methylene blue served as electronacceptor in the oxidative decarboxylation ofpyruvate.The results of the experiment shown in figure

9 suggest that the inhibition of hydrogen evolu-tion from pyruvate by oxidized methylene blueis due to its effect on the enzyme hydrogenase.To obtain further information on this point, theeffect of methylene blue on deuterium exchangewas determined in the presence and absence ofpyruvate. Such an experiment is illustrated infigure 10. The experimental conditions were

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essentially the same as those in the experimentshown in figure 9 except that exchange activityinstead of hydrogen evolution was measured.Curve A Mustrates deuterium exchange with thecell suspension only. As has been shown before,M/250 methylene blue inhibited the exchangereaction (curve B). The methylene blue remainedin the oxidized form. Curve C illustrates exchangein the presence of M/200 pyruvate. A smallincrease in the rate of exchange occurred, as wasnormally the case with relatively active cellsuspensions. Curve D Mustrates the effect ofadding methylene blue in a final concentration ofm/250 to the cell suspension five minutes beforeaddition of pyruvate. At the time of the firstreading (64 minutes) the methylene blue wasreduced. The following readings showed activeexchange, indicating that the inhibitory actionof methylene blue disappeared on conversion tothe leuco form. The concentration of methyleneblue and pyruvate used was the same as thoseemployed in the Warburg experiment shown infigure 9. Since hydrogen evolution from pyruvate

14

1

0 40 80 120 160 200 240MINUTES

Figure 10. The effect of pyruvate on methyleneblue inhibition of deuterium exchange.

A. Cell suspension onlyB. Cell suspension plus M/250 methylene blueC. Cell suspension plus m/200 pyruvateD. Cell suspension plus M/250 methylene blue

plus M/200 pyruvate

and exchange activity with deuterium both beginat the instant the methylene blue is reduced, itis logical to believe that the same enzyme-i.e.,hydrogenase-is involved in both cases.

DISCUSSION AND SUMMARY

It seems justifiable to conclude that catalysisof the exchange reaction between D2 and water isa property of the enzyme hydrogenase, and thatthis property is more fundamental than thevarious catalytic hydrogenations which arecustomarily employed for the measurement ofhydrogenase activity. Micrococcus aerogenes,which produces molecular hydrogen frompyruvate and certain other substrates, catalyzesthe exchange reaction even though unable tocatalyze hydrogenations of oxygen, ferricyanide,nitrate, fumarate, and methylene blue withmolecular hydrogen. These are potential hy-drogen acceptors commonly used for assay ofhydrogenase activity. The demonstration thatcelLs of M. aerogenes can hydrogenate artificialacceptors, such as methyl violet and benzylviologen, shows that an enzyme is presentwhich has the classical properties of hydrogenase,i.e., is able to carry out enzymatic activation ofmolecular hydrogen for the reduction of acceptorcompounds. Demonstration of hydrogenase inM. aerogenes by hydrogenation procedures aswell as by deuterium exchange should disposeeffectively of any argument that the exchangereaction may be due to nebulous hydrogenlyasesresponsible for evolution but not utilization ofmolecular hydrogen.Methylene blue in the oxidized form inhibited

not only the exchange reaction but also hy-drogenation of the acceptors benzyl viologen andmethyl violet. This toxicity of methylene blueexplains the failure to obtain evidence of hy-drogenase activity by hydrogen uptake withmethylene blue, both in the Thunberg test andin the Warburg method of assay. The strikinginhibition of hydrogenase in M. aerogenes bymethylene blue contrasts sharply with thesituation in most other bacteria where methyleneblue is the preferred hydrogen acceptor in assayof hydrogenase.Pyruvate was found to stimulate the exchange

activity of cell suspensions and extracts of M.aerogenes. This stimulation was most markedwith extracts which had been inactivatedpartially during the course of preparation. Benzyl

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viologen also stimulated exchange activity butto a lemser degree.Hydrogen production from pyruvate was

blocked by methylene blue in the oxidized form.Once the methylene blue was reduced, hydrogenwas evolved from pyruvate. Deuterium exchangein the presence of pyruvate also was blocked bymethylene blue in the oxidized form, but againonce the methylene blue was reduced, activeexchange occurred.

It has been established that methylene bluereversibly inactivates the hydrogenase in M.aerogene8, and hence the reduction of methyleneblue in the experiments with pyruvate must bedue to reducing systems other than molecularhydrogen, i.e., pyruvate, or a two-carbonfragment originating by decarboxylation ofpyruvate.The observation that both deuterium exchange

and the evolution of hydrogen from pyruvatebegin when the methylene blue is reduced makesit likely that the same enzyme is the criticalelement in both cases. This provides evidencethat hydrogenase is a necessary component ofthe enzyme complex responsible for the evolutionof molecular hydrogen from pyruvate as well asbeing involved in the exchange reaction and inthe hydrogenation of benzyl viologen and methylviolet.

REFERENCESDELEY, J. 1951 Metabolism of formate, bi-

carbonate and molecular hydrogen in nitrogendeficient E. coli. Enzymologia, 14, 296-303.

FARKAs, A. 1936 The mechanism of thecatalytic exchange reaction between deu-terium and water. Trans. Faraday Soc., 32,922-936.

FAnKAs, L., AND FIsCHER, E. 1947 On theactivation of molecular hydrogen by Protewvulgari. J. Biol. Chem., 167, 787-805.

GzST, H. 1952 Molecular hydrogen: oxidationand formation in cell free systems, pp. 522-543. In Phosphorus metabolism. Vol. II.Edited by W. D. McElroy and B. Glass. TheJohns Hopkins Press, Baltimore, Md.

GRUNBERG-MANAGO, M., SZULMAJSTER, J., ANDPROUVOST, A. 1951 Hydrogenlyase, formico-dehydrogenase et hydrogenase chez Escher-ichia coli. Compt. rend, 233, 1690-1692.

HOBERMAN, H. D., AND RITTENBERG, D. 1943Biological catalysis of the exchange reaction

between water and hydrogen. J. Biol. Chem.,147, 211-227.

HYNDMAN, L. A., BuRsS, R. H., AND WILSON,P. W. 1953 Properties of hydrogenase fromAzotobactor vinelandii. J. Bacteriol., 65, 522-531.

JOHNSTON, J., AND FRENKEL, A. 1951 Hy-drogenase activity of resting cultures ofRhodospirillum rubrum. Abstract of paper,Program of the American Society of PlantPhysiologists, Sept. 9, 1951, Minneapolis,Minn.

KOEPSELL, H. J., AND JOHNSON, M. J. 1942Dissimilation of pyruvic acid by cell freepreparations of Clostridium butylicum. J.Biol. Chem., 145, 379-86.

LICHsErIN, H. C., AND BoYD, R. R. 1953 Theformic hydrogenlyase system of Aerobacteraerogenes. J. Bacteriol., 65, 617-618.

McILWAN, H. 1948 Preparation of cell freebacterial extracts with powdered alumina.J. Gen. Microbiol., 2, 288-291.

ORDAL, E. J., AND HALVORSON, H. 0. 1939 Acomparison of hydrogen production fromsugars and formic acid by normal and variantstrains of Escherichia coli. J. Bacteriol., 38,199-220.

ORDAL, E. J., AND TsucHIYA, H. M. 1940 Theeffect of induced variation on fermentationsby E8cherichia communior. Proceedings,Third International Congress for Micro-biology, pp. 217-219, New York.

PAKES, W. W. C., AND JOLLYMAN, W. H. 1901The bacterial decomposition of formic acidinto carbon dioxide and hydrogen. J. Chem.Soc., 79, 386-391.

SMITH, G. N., AND MARSHALL, R. 0. 1952Fixation of molecular tritium by bacterialsuspensions. Arch. Biochem. and Biophys.,39, 395-405.

STADTMAN, E. R., NOVELLI, G. D., AND LIPMANN,F. 1951 Coenzyme A function in and acetyltransfer by the phosphotransacetylase sys-tem. J. Biol. Chem., 191, 365-376.

STEPHENSON, M., AND STICKLAND, L. H. 1931Hydrogenase, a bacterial enzyme activatingmolecular hydrogen. I. The properties ofthe enzyme. Biochem. J. (London), 25, 205-214.

STEPHENSON, M., AND STCKLAND, L. H. 1932Hydrogenlyases. Bacterial enzymes liber-ating molecular hydrogen. Biochem. J.(London), 36, 712-724.

TAUSS, J., AND DONATH, P. 1930 t'ber dieOxydation des Wasserstoffes und der Kohlen-wasserstoffe mittels Bakterien. Hoppe-Seyler's Z. physiol. Chem., 190, 141-168.

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