systems from different microorganisms, elec

A NEW PROCEDURE FOR ASSAY OF BACTERIAL HYDROGENASES1

HARRY D. PECK, JR.' AND HOWARD GESTDepartment of Microbiology, School of Medicine, Western Reserve University, Cleveland, Ohio

Received for publication June 17, 1955

Hydrogenase catalyzes the reversible oxida-tion of molecular hydrogen according to theformal reaction:

H2, 2H+ + 2e

and certain microorganisms which contain thisenzyme catalyze an exchange reaction betweenisotopic water and molecular gas (Hobermanand Rittenberg, 1943). This exchange activity isapparently paralleled by ability to interconvertortho- and para-hydrogen and both of thesereactions have been considered to represent"primary" assays for hydrogenase (Krasna andRittenberg, 1954; Fisher et al., 1954). In ordinarypractice, the enzyme is assayed by manometricmeasurement of H2 utilization in the presence of asuitable electron acceptor, usually an oxidation-reduction dye (Gest, 1954). Since hydrogenase isconcerned with hydrogen fomation in the normalmetabolism of a number of microorganisms, ithas been suggested (Gest, 1954) that under properconditions the evolution of H2 might be a moresuitable measure of enzyme activity. The presentpaper describes a simple manometric assay forhydrogenase based on formation of H2 from thereduced form of the dye methyl viologen, andapplications of this procedure to some problemsencountered in the study of hydrogen metabo-lism. The "evolution" procedure offers certaintechnical advantages and, in conjunction withother assay methods, should be of value forfurther research on the identity of hydrogenasesystems from different microorganisms, elec-tron transport pathways, and for purificationpurposes.

1 This investigation was supported by a grant(Contract No. AT(30-1)-1050) from the AtomicEnergy Commission. A preliminary report ofthis work was presented at the annual meetingof the Society of American Bacteriologists, NewYork, May 1955.

' Predoctorate Fellow of the National ScienceFoundation.

MATERIALS AND METHODS

Cultivation of organims and preparation of cell-free systems. Escherichia coli (Crookes strain;American Type Culture Collection 8739) andSerratia marcescens were grown at 37 C and roomtemperature, respectively, in deep stationaryculture (10 L in a 12-L Florence flask) in amedium containing glucose, 1 per cent; yeast ex-tract, 0.2 per cent; peptone, 0.2 per cent; nutrientbroth, 0.8 per cent; KH2PO4, 1.4 per cent; andNa2HPO4, 1.4 per cent. The non-gas-producingcoli-aerogenes variants WR 1, 2, 3, 5 and 6(for complete description see Gest and Peck,1955) and Proteu vulgaris OX-19 were similarlygrown (37 C) in a medium of the following com-position: glucose, 1 per cent; yeast extract, 1 percent; and tryptone, 1 per cent.

Clostridium butylicum (Northern Regional Re-search Laboratory No. B-593) and Clostridiumpasteurianum (strain W5) were cultivated at 37C in the complex glucose medium described byWilson et al. (1948), while Micrococcus lactilyticus(strain 221) was grown (37 C) in the lactate me-dium used by Whitely (1953). With the lattertwo bacteria, stationary 10-L cultures were em-ployed as noted previously. In order to achievethe strict anaerobiosis required for growth of C.butylicum, the freshly autoclaved and rapidlycooled medium was inoculated; the flasks werethen gassed with sterile helium or nitrogen, andsealed off using a mercury valve. Complete re-moval of oxygen was also necessary for success-ful cultivation of Desulfovibrio desulfuricans(Hildenborough strain); this organism was grownat 30 C in a lactate medium suggested by Dr. J.R. Postgate (very similar to the medium B de-scribed by Butlin et al., 1949). The freshly de-oxygenated lactate medium was sown with alarge inoculum. The flask was filled almost to thetop with additional medium, and the culture wassealed with a sterile rubber stopper; the latterbore a glass tube which connected with a trap(small Erlenmeyer) containing a solution of

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NEW HYDROGENASE ASSAY PROCEDURE

alkaline pyrogallol. The C. butyricum preparationwas a lyophilized extract kindly supplied by Dr.R. S. Wolfe. We are also indebted to Dr. HenryKoffler for fresh extracts of Lactobacillu del-bueckii.

Azotobacter vinelandii (strain 0) was grown at30 C with vigorous aeration in a nitrogen-freesucrose medium (Burk et al., 1934); Rhizobiummeliloti (No. 103 Wisconsin) was grown at thesame temperature on an agar medium contain-ing sucrose, an ammonium and other salts (asused by Wilson and Wilson, 1942), and 400 mgyeast extract per L.

Rhodospirillum rubrum (strain SI) was grownphotosynthetically in a malate plus glutamatemedium (G3X medium) according to the pro-cedure of Kohnmiller and Gest (1951).In all instances an effort was made to harvest

the cells (usually with a Sharples centrifuge)during a period of active growth. C. butylicumcell paste was ground with 60-mesh glass powder(2.5 g per g wet weight of cells) and extracted withdistilled water or 0.05 M phosphate buffer pH6.5 (1.5 ml per g of cell paste used). Alumina A301 was the grinding agent for all of the otherorganisms. To obtain the particulate cell-freehydrogenase of A. vinelandii, the procedure out-lined by Hyndman et al. (1953) was followed. Theother organisms were ground with 2.5 g of alu-mina per g wet weight of celLs, and the mixtureextracted with 1.5 ml of water per g of cell paste.Ordinarily, the suspension at this stage wascentrifuged for 10 min at 16,000 xG and thesupernatant fluid further clarified by centrifuga-tion for 1 hr at 20,000 XG. Extracts and othercell-free preparations were dispensed in tubes;the latter were flushed with hydrogen (or he-lium), stoppered, and stored at -20 C.

Special reagents. Benzyl viologen was obtainedfrom British Drug Houses, Ltd., Toronto,Canada, and methyl viologen from JacobsonVan Den Berg and Co., 73 Cheapside, LondonE.C. 2, England.

REsUIs

Principle of the Assay. Reversibility of hydro-genase action is indicated by the observationthat preparations containing the enzyme cata-lyze the ortho-para interconversion and theisotopic exchange reaction between gas andwater. Furthermore, potentiometric measure-ments have shown that intact cells of E. coli,

containing hydrogenase, catalyze the reaction

H2 = 2H+ + 2e

in accord with the classical hydrogen elec-trode equation, provided methyl viologen isadded as a mediator (Green and Stickland, 1934).Methyl viologen is one of a family of dyes, de-

scribed by Michaelis and Hill (1933), with thegeneral structure shown in figure 1.These indicators differ from most other oxida-

tion-reduction dyes in several important respects.They are colorless in the oxidized state, while thereduced forms exhibit a deep blue or violet color.The stable reduced form under physiologicalconditions (pH < 12) is generated by the addi-tion of one electron. At 30 C, the normal poten-tial of methyl viologen is -0.446 volts, and thepotential of the system is independent of pH(Michaelis and Hill, 1933). Since the potentialof the hydrogen electrode decreases with in-creasing pH, the potential of the dye systemunder alkaline conditions is more positive thanthat of the hydrogen electrode, whereas inacid solution it is more negative than that ofthe hydrogen electrode at the same pH. Itwould be expected, therefore, that molecular hy-drogen would be evolved in acid solution fromthe reduced form of the dye in the presenceof a suitable catalyst such as palladium orplatinum. Such a reaction was observed byMichaelis and Hill (1933), who used chromouschloride as the reductant for methyl viologenand colloidal Pd as the catalyst. Assuming thathydrogenase acts catalytically in a similar man-ner, formation of H2 from a reservoir of reducedmethyl viologen would be anticipated. In theprocedure described below, H2 is produced bythe enzyme system from methyl viologen main-

OXIDIZED FORM

R

+NC H

-C0HC I N CH

1 1NC CH

R

R - BENZYL ORMETHYL GROUP

REDUCED FORM

R

II IN CN11 11 \sCz

oCs11 1HC CH

IR

Figure 1. Structure of viologen dyes

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tained in the reduced state by an excess of thestrong reducing agent sodium hydrosulfite.

Assay Procedure. The hydrogenase prepara-tion, together with buffer of appropriate pH(total volume, 0.8 ml), is placed in the mainchamber of a Warburg vessel of approximately10-ml capacity. 0.1 ml of 20 per cent KOH isplaced in the center well (with ifiter paper) and0.2 ml of methyl viologen solution (80 Am/ml)in the side arm bearing the solid plug. The vesselis then attached to the manometer and flushed(outside the Warburg bath) with helium (ornitrogen) for 10 to 15 min.Two ml of 0 125 N NaOH are added to 3 ml of

water and, just before use, 210 mg of sodiumhydrosulfite are dissolved in the solution. 0.2ml of the hydrosulfite reagent is quickly pipettedinto the side arm containing the methyl viologen(while the vessel is being gassed). After severalminutes, the vessels are closed and equilibratedin the bath (30 C) in the usual manner. At zerotime, the hydrosulfite + methyl viologen solu-tion is added to the mixture in the main chamber.The amount of alkali required to attain a

neutral pH (6.5 to 7) varies, depending on thebatch of hydrosulfite used, and must be deter-mined empirically. Since the pH of the neutralizedhydrosulfite solution falLs rapidly in the presenceof air, the reagent should be added to the dye inthe side arm as quickly as possible. Failure tostandardize this step may cause difficulty incontrol of the pH of the reaction mixture. Thesolid hydrosulfite stock should be protected fromdeterioration by atmospheric oxidation; this is

TABLE 1Requirements for the evolution assay

pL HgFlask Contents Evolved

per 10 Min

Hydrosulfite + methyl viologen....... 1Extract + hydrosulfite................ 0Extract + methyl viologen............ 1Extract + hydrosulfite + methylviologen............................ 50

Extract (boiled) + hydrosulfite +methyl viologen..................... 2

* In all instances, the buffer was 0.0625 M po-tassium phosphate pH 6.5. Where indicated, 16Am of methyl viologen, 40 im of sodium hydro-sulfite, and 0.001 ml of Clostridium butylicumextract were used.

conveniently done by dispensing the solid intoscrew-cap test tubes which are briefly flushed outwith a stream of helium after each use.

Results of a typical test of the assay procedure,using C. butylicum extract as a source of hydro-genase, are shown in table 1.

It is evident that gas evolution occurs only inthe presence of reduced methyl viologen andhydrogenase. Hydrogen evolution is not ob-served with boiled extracts or when eitheroxidized methyl viologen or hydrosulfite alone isadded.3 Similarly, gas is not produced underproper assay conditions by extracts from or-ganisms which do not manifest some type ofhydrogen metabolism (see below). At enzymelevels which show a relatively high rate of H2production (approx. 50 ,L/10 min) with reducedmethyl viologen, virtually no evolution is ob-served with hydrosulfite plus benzyl viologen(Eo' = -0.359), rosinduline G (Eo' - -0.281),or methylene blue (Eo' - +0.011). Benzylviologen does show some activity as a mediatorat higher enzyme concentrations.

Analysis of Assay Conditions. Identificationof the gas evolved. The gas produced was alkaliinsoluble, thus eliminating carbon dioxide andsome possible degradation products of hydro-sulfite such as H2S, SO2 and SOs. The usualmanometric methods for identification of hydro-gen are based on reduction of a dye in the pres-ence of catalysts such as platinum black orpalladinized asbestos (Cardon and Barker,1947; Gest et al., 1950). In our hands, suchcatalysts effected a very slow absorption of H2,possibly because the quantities of gas involvedwere very small. A crude extract from C. pas-teurianum, containing hydrogenase active withmethylene blue, was therefore used as the catalystas shown in figure 2.A C. butylicum extract was allowed to evolve

gas from methyl viologen and a limiting quan-tity of hydrosulfite (curve A). When the produc-tion of gas had ceased (in a duplicate vessel), asolution of methylene blue plus C. pasteurianumhydrogenase was added from a side arm, causinga utilization of gas which approached the amountoriginally evolved (curve B). If the methyleneblue is carefully layered over the C. pasteurianum

'When overwhelming amounts of certain hy-drogenase preparations are used, a relativelysmall evolution of H2 may be observed with hy-drosulfite alone.

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100 -0

A

75 -

50

-I

25-

030 60 90 120

MINUTESFigure B. Identification of the gas evolved from

reduced methyl viologen. Curve A represents thecourse of gas production by 0.01 ml of Clostridiumbutylicum extract in 0.0625 M phosphate pH 7.0 inthe presence of 8 pM of sodium hydrosulfite and4.8 Am of methyl viologen. In a similar vessel(curve B), 0.2 ml of Clostridium pasteurianumhydrogenase (crude extract) and 12 um of meth-ylene blue were added from the side arm at thetime indicated by the arrow.

preparation in the side arm, there is no appre-ciable gas utilization until these materials areadded to the main chamber. Reabsorption ofgas occurred only when both C. pasteurianumhydrogenase and electron acceptor were added.Since C. butylicum extract does not activelyreduce methylene blue with H2, there is no sig-nificant gas reabsorption unless C. pasteurianumextract is used.On the basis of the foregoing tests, we con-

clude that the gas evolved from methyl viologenand hydrosulfite is molecular hydrogen.

Relaionship between activity and enzyme con-centration. The rate of hydrogen evolution isdirectly proportional to enzyme concentrationover a limited range as imustrated in figure 3.

It may be noted that, under the conditionsemployed, a slight break in the curve is evidentwhen the rate is approximately 75 MAL per 10

CO

z

0120

a.

> 080

III

ea=

1 2 3 4MILLILITERS OF EXTRACT (x10)

Figure 3. Relationship between enzyme con-centration and hydrogen evolution activity. Acrude extract of Clostridium butylicum was em-ployed using the conditions described underAssay Procedure. Buffer: 0.0625 M potassiumphosphate pH 6.5.

min. The point at which this deviation occurs isaltered by changing the concentration of eitherhydrosulfite or methyl viologen. Since bettermanometric accuracy is achieved with lowerrates, the concentration of extract used in assay-ing preparations was chosen so as to give valuesin the range of 40 to 60 ,uL per 10 min. In thisrange of activity, the rates of hydrogen forma-tion were constant for about twenty minutes.

Optimal concentrations of methyl viologen andhydrosulite. Figure 4 summarizes the influence ofvarying methyl viologen and sodium hydro-sulfite concentrations on the rate of H2 formationusing a constant amount of C. butylicum extract.As indicated, as much as 60 Mm of hydrosulfite

per 1.2 ml may be used. Methyl viologen, how-ever, is inhibitory at concentrations greater than16 ,MM per 1.2 ml. In order to obtain maximalrates, 40 umM of sodium hydrosulfite and 16 Mmof methyl viologen were employed in routineassays.

Influence of pH on hydrogenase activity. The

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-O 20 40 60 0 8 I6 24M OF SOIUM uMOF METYHYDROSULITE VIOL.OGEN

Figure 4. Optimal concentrations of sodiumhydrosulfite and methyl viologen in the evolu-tion assay. Conditions as described under AssayProcedure, except for the experimental variablebeing tested. Buffer: 0.0625 M potassium phos-phate pH 6.5.

effects of pH on evolution activity and reductionof methylene blue by H2 were studied using ex-tracts of M. lactilytius, C. butylicum, and E.coli. With the preparation from M. lactilytc,evolution from reduced methyl viologen exhibiteda narrow optimum at about pH 6.3, whereasmethylene blue reduction was optimal at pH7.3. Similar results were observed using extractsfrom anaerobically-grown E. coli (Crookes).Although the pH optimum for methylene bluereduction by hydrogenases from various bac-tetia is generally at a slightly alkaline value, anoptimum of 6 to 6.3 has been reported for thisreaction in M. lactilytibus extracts (VVitter,1953).Optimal evolution activity in C. butylicum

extracts occurred at a pH of 7. In this connec-tion it should be remarked, however, that evolu-tion of H2 in cell-free systems from pyruvate bythe phosphoroclastic reaction (Koepsell andJohnson, 1942), and from formate by the hydro-genlyase reaction (Gest, 1952b), is ma ima onthe acid side.

Asuigthat hydrogenase acts as a hydrogenelectrode according to the reaction previouslynoted, it would be expected that evolution ac-tivrity would be favored by incraed H+ concen-tration, whereas oxidation of H2 with electronacceptors such as methylene blue would occurmore readily at decreased H+ concentrations.

Effects of a hydrogen atmosphere on evolutinacfiiy. Depending on the substrate and theorganism employed, various degrees of inhibition

of H2 production by an atmosphere of molecularhydrogen have been reported (Gest, 1954). Itseemed possible that the variations observedmight be primarily due to differences in reversi-bility of the initial electron-donating reactionsand, accordingly, that a more consistent in-hibition pattern might be revealed in studiesusing the evolution of H2 from reduced viologenas the assay.The rate of hydrogen evolution from reduced

methyl viologen was the same whether helium ornitrogen was used as the gas phase. When theatmosphere was hydrogen, however, evolutionactivity was depressed. Representative resultsusing three types of preparations are given intable 2, which also summarizes the effects of ahydrogen gas phase on H2 formation from physi-ological precursors.

It is clear that the degree of inhibition of evolu-tion activity from reduced viologen was not thesame in the preparations tested and also thatthere are marked differences when the electrondonor is reduced methyl viologen as comparedwith formate or pyruvate. In contrast to theresults of Lipmann and Tuttle (1945), productionof H2 by our extracts of C. butylicum was virtuallynot inhibited by a hydrogen gas phase. The otherresults noted using pyruvate and formate are in

TABLE 2Inhibition of H2 evolution by an atmosphere of

molecular hydrogen

Per Cent Inhibition ofH2 Evolution from:

Extract RePyru- For- ducedvate matet vio-

logent

Escherichia coli.............. 33 59Micrococcus lactilyticus...... 41 _ 87Clostridium butylicum........ 4 24

The final fluid volume in the vessels was 1.2 ml.Each center well contained 0.1 ml of 20 per centKOH. Controls were simultaneously run usinghelium as the gas phase.

* 0.1 ml of extract in 0.0625 M phosphate pH6.3; 100 Am sodium pyruvate.

t 0.2 ml of extract in 0.0625 M phosphate pH6.0; 60 Am sodium formate.

t Conditions as described under Assay pro-cedure, using 0.0625 M phosphate pH 7 and 0.03ml E. coli extract, 0.001 ml M. lactilyticus extract,or 0.001 ml of C. butylicum extract.

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TABLE 3Distribution of hydrogenase as indicated by the evolution and methylene blue reduction assays

The preparations listed contained different concentrations of protein; the relative values for the twoassays can be compared quantitatively for a given organism but only qualitative comparisons are pos-sible among the different preparations. Extracts were used in all instances except as indicated.

Activity by Activity by Abjility to ProducePreparation Methylene Blue Evolution Assayt H from Formate

Reduction Assay* and/or Pyruvate

juL H, utilzed/hr/mi pL H2 evolved/hr/lmEscherichia coli ..................................... 9,000 14,400 +Proteus vulgaris..................................... 25,500 4,200 +Variant WR I ....................................... 12,200 3,360 +Variant WR 2....................................... 13,800 6,900 -

Variant WR 3....................................... 0 0 -

Variant WR 5....................................... 11,000 1,860 -

Variant WR 6....................................... 0 0 -

Serratia marcescens.................................. 0 0 -

Clostridium butylicum............................... 0 48,000 +C. pasteurianum..................................... 10,200 12,000 +Micrococcus lactilyticus.............................. 75,600 112,800 +Desulfovibrio desulfuricans-extract ....... ........... 101,300 57,400 +D. desulfuricans-intact cells........................ 40,000 47,000 +Azotobacter vinelandii-extract ........ ............... 3,420 0 -

A. vinelandii-intact cells........................... 2,190 0 -

Rhizobium meliloti-intact cells...................... 0 0 -

Rhodospirillum rubrum ............. ................. 1,110 0 -

*0.0625 M phosphate pH 7.3; 8 im methylene blue; 0.1 ml 20 per cent KOH in center well; final fluidvolume, 1.2 ml; temperature, 30 C; gas phase, hydrogen.

t Conditions as described under Assay procedure, using 0.0625 M phosphate pH 7.

good agreement with values previously reported(Gest, 1952b; Witter, 1953).

It is of interest that evolution activity fromreduced viologen and from pyruvate or formatewas inhibited to the greatest extent in the ex-

tracts of M. lactilyticus and to the smallest ex-

tent in the C. butylicum preparation. Also sig-nificant is the finding that the inhibition is

invariably much greater with reduced viologenserving as electron donor than with the physi-ological substrates.The explanation for the quantitative differences

described is not apparent at present, and it ispossible that the effects noted may be complicatedto some degree by the efficacy of H2 in reversing

the inactivation of "oxidized" hydrogenase(Back et al., 1946; Joklik, 1950; Gest, 1952a;Fisher et al., 1954). Thus we have observed incertain preparations from E. coli an actual 30to 50 per cent increase in the rate of hydrogenproduction from formate when the reaction was

measured under an atmosphere of hydrogenrather than under helium.

Judging from the results in table 2, the forma-tion of H2 from reduced viologen is a reversibleprocess. This conclusion is also supported by theobservation that all preparations capable ofevolving hydrogen from reduced methyl viologenalso can oxidize H2 with benzyl viologen as theelectron acceptor.'

Occurrence of "evolution" and "methylene blue-reduction" activities among microorganim. Prepa-rations from a variety of bacteria were tested forability to evolve H2 from reduced methyl viologenand also for capacity to oxidize molecular hy-drogen in the presence of methylene blue. Theresults of this survey are given in table 3. Fromthe data given it is evident that most of thepreparations showed both types of activity or

neither. There are, however, several noteworthyexceptions which are discussed below.

Extracts of C. butylicum (and the very similarC. butyricum) prepared by the usual procedures4Benzyl viologen is more suitable (than the

methyl dye) as an electron acceptor because of itshigher oxidation-reduction potential.

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aw

N

s=o /Ct'

z

0

v.30

0

0 10 20 30MINUTES

Figure 5. Oxidation of H2 by Clostridiumbutylicum extract with benzyl viologen as elec-tron acceptor. At zero time, 16 pM of benzylviologen were added to 0.15 ml of extract in 0.04m veronal buffer pH 7.6. Final fluid volume, 1.2ml; 0.1 ml of 20 per cent KOH in center well; gas

phase, H2; temp, 30 C.

rapidly produce H2 from pyruvate but do notactively reduce methylene blue under a hydrogenatmosphere (Koepsell and Johnson, 1942; Wolfeand O'Kane, 1953).6 The latter observation hasbeen frequently interpreted as evidence that a

typical hydrogenase is not present in suchpreparations. The enzyme is obviously present,as indicated by the evolution assay and by thecapacity of such extracts to reduce benzylviologen with molecular hydrogen under certainconditions (Gest, 1954; Peck and Gest, 1954a)(see figure 5).The rate of benzyl viologen reduction is not

linear with time. The induction period suggeststhat the oxidized form of the dye is relatively in-hibitory, or that this particular hydrogenase is

maximally active only when a low redox potentialis attained (see also Discussion). This activity

G Similar observations with M. aerogenes were

recently reported by Curtis and Ordal (1954),who also observed that H, is utilized by thesepreparations in the presence of benzyl viologenor methyl violet.

appears to be quite labile with respect to inac-tivation by oxygen.Attempts to demonstrate significant oxidation

of H2 with methylene blue in extracts of C.butylicum yielded uniformly negative results. Oc-casional extract preparations effected an ephem-eral reduction of this dye, but only when thehydrogenase was kept in the concentrated formin the side arm and added to the methylene bluein the main chamber at zero time. In table 4,the relative hydrogenase activities observed inan extract of C. butyricum using different asayprocedures are compared.The rate of H2 evolution observed with reduced

methyl viologen is more than sufficient to ac-count for the rate of hydrogen production frompyruvate and is in fact approximately 10 timesas great (per mg of extract). Similar results wereobtained using extracts from C. butylicum. Underthe conditions noted in the table (pH 6.5 andfinal fluid volume 3 ml), 12 was not oxidizedwith methylene blue or benzyl viologen. Usingthe same preparation, however, at a concentra-tion of 17 mg per ml and at pH 7.5 there was arelatively weak consumption of H2 with benzylviologen and very weak activity with methyleneblue.One or more components of the multienzyme

TABLE 4Hydrogenase activity in Clostridium butyricum

extract

bpi H2Flask Contents' Gas Phase per 10

Mn.

Extract (10 mg) +88m meth-ylene blue................. Hydrogen 0

Extract (10 mg) + 16 pum ben-zyl viologen.. Hydrogen 0

Extract (10 mg) + 100 oMsodium pyruvate........... Helium 140

Extract (2mg) + hydrosulfite+ methyl viologen ......... Helium 316

Extract (2mg) + hydrosulfite. Helium 36Extract (boiled; 10mg) + hy-

drosulfite + methyl vi-ologen.. Helium 20

* Experimental conditions: 0.033 M phosphatepH 6.5; final fluid volume of 3.0 ml in vessels of25-ml capacity; 0.2 ml of 20 per cent KOH incenter well. The hydrosulfite and methyl viologenwere used as described under Assay procedure.

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system responsible for H2 formation from pyru-vate in clostridial extracts is very labile (Wolfeand O'Kane, 1953). Preparations which havelost the ability to evolve hydrogen from pyruvate,however, still show high levels of hydrogenase as

measured by gas evolution from reduced methylviologen.

Preparations which reduce methylene blue but donot show evolution activity. Three preparations ofthis kind were disclosed by the survey and re-lated experiments. As indicated in table 3, ex-tracts from the photosynthetic bacterium R.

rubrum showed weak hydrogenase activity withmethylene blue but did not produce H2 from re-duced viologen (the benzyl dye was also not re-duced by the preparation in the presence of H2).Illumination at an intensity of approximately1,000 foot candles had no effects. Intact celLs ofR. rubrum produce H2 photochemically (Gest etal., 1950) but this activity is absent from theextracts prepared thus far.The particulate hydrogenase preparation from

A. vinelandii showed the same qualitative be-havior as the Rhodospirillum extract (table 3).Since these results suggested the possibility thatevolution and reduction (methylene blue) ac-tivities might be attributable to two differenthydrogenases, or to a single hydrogenase withtwo prosthetic groups, attempts were made toseparate the two types of activity from extractsof M. lactilyticu.Protamine (0.3 volume of a 2 per cent solution)

was added to the M. lactilyticus extract (pre-pared as described in Materials and Methods)and the resulting precipitate removed by cen-trifugation. Three volumes of saturated ammo-nium sulfate, pH 6.5, were then added and theresulting precipitate suspended in % the originalvolume of 0.05 M phosphate buffer (pH 6.5) con-

taining 0.01 per cent cysteine. Saturated am-

monium sulfate was added to this solution (un-dialyzed) until a faint precipitate appeared; thequantity of ammonium sulfate necessary for thisstep varied in different fractionations. Theprecipitate was collected and suspended in thephosphate + cysteine mixture (Ko the originalvolume). As shown in table 5, this fraction (whichwas slightly turbid and probably particulate)showed virtually no ability to evolve gas fromreduced methyl viologen or to reduce the benzyldye with H2, but it did reduce methylene blue.

Recent experiments of Shug et al. (1954) indi-

TABLE SSeparation of hydrogenase activities byfractionation

The data are given as aL H2 utilized or evolvedper ml per 10 minutes.

AmmoniumAssay Extract SulfateEtatFraction

Methylene blue reduction ... 6,800 1,560Benzyl viologen reduction... 1,150 40Evolution from reducedmethyl viologen............ 12,200 100

For the reduction assays, 16pM of benzyl violo-gen or 8 pM of methylene blue were used underthe conditions noted for table 3. Evolution ac-tivity was assayed as described previously.

cate that MoO3 and flavin adenine dinucleotideare required for reduction (by H2) of "one-electron" acceptors such as cytochrome c bypreparations from C. pasteurianum. Additionof the former materials or boiled extracts to thefractionated M. lactilyticus hydrogenase (in phos-phate buffer) did not induce hydrogen evolutionfrom reduced methyl viologen. Further at-tempts to evoke evolution activity by addingother protein fractions (devoid of hydrogenaseactivity) from the M. lactilyticus extract met withfailure. The hydrogenase fraction which onlyreduced methylene blue was very stable tostorage at -20 C, while the soluble hydrogenaseactivity remaining in the supernatant from thefirst ammonium sulfate precipitation decayedrapidly. The soluble fraction did not utilize H,in the presence of ferricyanide, whereas the"particulate" fraction reduced this acceptorreadily.

DISCUSSION

Fisher et al. (1954) assume that the isotopicexchange reaction between gas and water is adirect measure of the primary reaction catalyzedby hydrogenase. Purification of hydrogenaseusing exchange activity as the assay has notbeen studied, however, and there is practicallyno data available comparing the ratio of exchange(or ortho-para conversion) activity with anyother manifestation of hydrogenase action indifferent microbial preparations. Furthermore,a recent report suggests that the exchange ac-tivity of certain preparations is surprisingly slowas compared with rates of dye reduction (Hynd-man et al., 1953). With regard to sensitwity of

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the exchange assay, this may well be less sensitivethan assays based on hydrogenation or evolution.Thus, exchange activity is markedly depressedupon addition of electron acceptors, particularlyin systems wherein the terminal enzymes are inexcess and pull the over-all reaction in favor ofreduction (e. g., see Farkas and Fischer, 1947).These considerations indicate the need for moreextensive study of the exchange reaction and theortho-para conversion.

Inherent in previous discussions of a properassay for hydrogenase is the assumption thatthe enzyme is in fact identical or very similar inall organisms. The present data suggest thatthere may be more than one type of hydrogenase,perhaps even in the same organism. An alterna-tive possibility is that hydrogenase may be a"double-headed" enzyme with two prostheticgroups. With the latter interpretation, oneprosthetic group might be considered to mediateelectron transport to dyes or physiological ac-ceptors of relatively high redox potential, whilethe other might be concerned with reduction of"one-electron" acceptors of low potential andalso with formation of molecular hydrogen. It issignificant that all of the preparations reputed tocontain hydrogenase (Gest, 1954) were found toreduce methylene blue or to produce H2 from re-duced methyl viologen (or both). Also, extractswhich produce H2 from physiological precursorssuch as formate or pyruvate invariably evolvehydrogen by the new assay procedure (see table3).

It is likely that clarification of the significanceof the various assay procedures employed here-tofore will necessitate purification of the hy-drogenase protein(s) from various sources. Oneof the major difficulties encountered in purifica-tion studies is the extreme sensitivity of the en-zyme to oxygen. The recent investigations ofFisher et al. (1954) indicate that hydrogenase isinactivated by both oxidation and oxygenation;the latter can be reversed by physical or chemicalremoval of oxygen. Oxidation inactivation isapparently caused not only by reaction withmolecular oxygen, but also in some instances bysubstances or systems which can act as terminalacceptors for oxidation of H2. We have observedan interesting situation of this kind in studies onfumarate reduction by H2 in the system:C. butylicum hydrogenase ... benzyl viologen ...

E. coli fumaric reductase

The reductase enzyme is obtained by extractionof an acetone-dried particulate fraction from E.coli with 1.5 M sodium chloride (Peck and Gest,1954a). The combination of C. butylicum hy-drogenase and reductase extract does not reducefumarate with H2 unless a catalytic mediator suchas benzyl viologen is added., Reaction rate isdirectly proportional to reductase concentrationonly if the reductase system is not present ingreat excess. When reductase is present at highconcentration, the viologen mediator remains inthe oxidized form and there is no utilization ofhydrogen. In other words, the reaction proceedsonly if the redox potential is such that there is asteady state concentration of reduced benzylviologen in the system.The exact mechanism by which viologens medi-

ate the evolution of H2 from hydrosulfite andreduction of acceptors such as fumarate (Peckand Gest, 1954a) and pyridine nucleotides (Peckand Gest, 1954b) is still unknown. Attempts toreplace methyl viologen in the evolution assay(using different hydrogenases) with di- or tri-phosphopyridine nucleotides, flavins, or metalshave thus far given negative results.

Considering the problem of inactivation due tooxygen or to oxidation by the electron acceptorsystem in the usual type of assay, the new evolu-tion procedure offers potential technical ad-vantages. The very powerful reducing mixtureof methyl viologen plus hydrosulfite unques-tionably removes all traces of oxygen from thesystem and, judging from the work of Fisher etal. (1954) and Joklik (1950), should be effectivein reversing, to some extent at least, inactivationwhich occurs during manipulations. The advan-tage this offers for purification purposes isobvious.A modification of the present procedure is the

use of a catalytic amount of methyl viologentogether with an enzyme system which willcontinuously reduce the dye. A formic dehydro-genase found in certain coli-aerogenes variants ac-tively reduces methyl viologen in the presence offormate and can be used as the hydrogen donorsystem in this manner (Gest and Peck, 1955). Thecomplete system in this case is a model formic

6 Fumarate reduction by molecular hydrogenin extracts of M. lactilyticu8 is similarly activatedby addition of benzyl viologen (Peck and Gest,1954a).

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hydrogenlyase complex which decomposes for-mate according to the equation:

HCOOH -- C02 + H2Attempts have been made to obtain a donorsystem which will furnish electrons to the hy-drogenases of the strict anaerobes (Desulfovibrio,Micrococcus, and Clostridium) in the absence of aviologen mediator. The formic dehydrogenasereferred to above can fulfill this function withcell-free hydrogenases from coli-aerogenes or-ganisms, but cannot couple with the correspond-ing enzyme in extracts of the strict anaerobes(Gest and Peck, 1955). Since the latter prepara-tions can produce H2 rapidly from pyruvate, thisobservation indicates that there must be an im-portant difference in the electron transport path-way in the phosphoroclastic and hydrogenlyasereactions. It should also be noted that we haveattempted to couple the pyruvate oxidase sys-tem of L. delbrueckii (Hager et al., 1954) withthe hydrogenases of C. butylicum, M. lactilyticusand E. coli, but without success.

Evaluation of the significance of the presentprocedure as a "primary" assay will requirepurification studies with soluble hydrogenasesand also more detailed examination of the threepreparations which reduce methylene blue butdo not evolve H2 from reduced viologen. Sincethe latter preparations appear to be particulatein nature, it is conceivable that there may be astereochemical or permeability barrier with re-spect to the reduced dye in these instances. Onthe other hand, intact cell suspensions of bac-teria readily produce hydrogen from reducedmethyl viologen in those cases where the solublecell-free system shows this activity. It is possiblethat the exchange reaction (and the ortho-paraconversion) is a "primary" assay, but until thisis conclusively shown it seems prudent to employreduction of dyes (also ferricyanide etc.) andevolution from reduced viologen as comparativeassay procedures. There is little doubt that use ofthe latter assays will also provide information ofvalue concerning the many possible pathways ofelectron transport in the oxidation and evolutionof molecular hydrogen (Gest, 1954).

ACKNOWLEDGMENTSThe authors are indebted to Dr. Joseph Judis

for testing various bacterial preparations and toMiss Marion A. Koser for valuable technicalassistance.

SUMMARY

A new manometric procedure for assay ofhydrogenase activity is described. The method isbased on measurement of molecular hydrogenevolved from the reduced form of the "one-electron" dye methyl viologen. Cell-free extractsand intact cells of a variety of microorganismswere examined for the presence of hydrogenaseas indicated by the evolution assay and bycapacity to oxidize molecular hydrogen in thepresence of methylene blue. All preparationsreputed to contain hydrogenase showed eitherone or both types of activities. The results of thesurvey and related experiments are discussed inrelation to current concepts of the mechanismof hydrogenase action and it is suggested that theevolution assay will be a valuable adjunct toother assay procedures in the further study ofenzymatic oxidation and formation of molecularhydrogen.

REFERENCESBACK, K. J. C., LASCELLES, J., AND STILL, J. L.

1946 Hydrogenase. Australian J. Sci. Re-search, 9, 25.

BURK, D., LINEWEAVER, H., AND HORNER, C. K.1934 The specific influence of acidity on themechanism of nitrogen fixation by Azoto-bacter. J. Bacteriol., 27, 325-340.

BUTLIN, K. R., ADAMS, M. E., AND THOMAS, M.1949 The isolation and cultivation of sul-phate-reducing bacteria. J. Gen. Microbiol.,3, 46-59.

CARDON, B. P. AND BARKER, H. A. 1947 Aminoacid fermentations by Clostridium propioni-cum and Diplococcus glycinophilus. Arch.Biochem., 12, 165-180.

CURTIS, W. AND ORDAL, E. J. 1954 Hydro-genase and hydrogen metabolism in Micro-coccus aerogene8. J. Bacteriol., 68, 351-361.

FARKAS, L. AND FISCHER, E. 1947 On theactivation of molecular hydrogen by Proteusvulgaris. J. Biol. Chem., 167, 787-805.

FISHER, H. F., KRASNA, A. I., AND RITTENBERG,D. 1954 The interaction of hydrogenasewith oxygen. J. Biol. Chem., 209, 569-578.

GEST, H. 1952a Properties of cell-free hy-drogenases of Escherichia coli and Rhodos-pirillum rubrum. J. Bacteriol., 63, 111-121.

GEST, H. 1952b Molecular hydrogen: oxidationand formation in cell-free systems. InPhosphorus Metabolism, pp. 522-543, Vol. II,edited by W. D. McElroy and B. Glass. TheJohns Hopkins Press, Baltimore, Md.

GEST, H. 1954 Oxidation and evolution of

19561 79


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molecular hydrogen by microorganisms.Bacteriol. Revs., 18, 43-73.

GEST, H., KAMEN, M. D., AND BREGOFF, H. M.1950 Studies on the metabolism of photo-synthetic bacteria. V. Photoproduction ofhydrogen and nitrogen fixation by Rhodo-spirillum rubrum. J. Biol. Chem., 182, 153-170.

GEST, H. AND PECK, H. D., JR. 1955 A studyof the hydrogenlyase reaction with systemsderived from normal and anerogenic coli-aerogenes bacteria. J. Bacteriol., 70, 326-334.

GRzEN, D. E. AND STICKLAND, L. H. 1934Studies on reversible dehydrogenase sys-tems. I. The reversibility of the hydro-genase system of Bact. coli. Biochem. J.(London), 28, 898-900.

HAGER, L. P., GELLER, D. M., AND LIPMANN, F.1954 Flavoprotein-catalyzed pyruvate oxi-dation in Lactobacillus delbrueckii. Federa-tion Proc., 13, 734-738.

HOBERMAN, H. D. AND RITTENBERG, D. 1943Biological catalysis of the exchange reactionbetween water and hydrogen. J. Biol.Chem., 147, 211-227.

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

JOKLIK, W. K. 1950 The hydrogenase of E.coli in the cell-free state. I. Concentration,properties and activation. Australian J.Exptl. Biol. Med. Sci. 28, 321-329.

KOEPSELL, H. J. AND JOHNSON, M. J. 1942Dissimilation of pyruvic acid by cell-freepreparations of Clostridium butylicum. J.Biol. Chem., 145, 379-386.

KOHLMILLER, E. F., JR. AND GEST, H. 1951 Acomparative study of the light and dark

fermentations of organic acids by Rhodo-spirillum rubrum. J. Bacteriol., 61, 269-282

KRASNA, A. I. AND RITTNBERG, D. 1954 Themechanism of action of the enzyme hydro-genase. J. Am. Chem. Soc., 76, 3015-3020.

LIPMANN, F. AND TUTTLE, L. C. 1945 On thecondensation of acetyl phosphate with for-mate or carbon dioxide in bacterial extracts.J. Biol. Chem., 158, 505-519.

MICHAELI, L. AND HILL, E. S. 1933 Theviologen indicators. J. Gen. Physiol., 16,859-873.

PECK, H. D., JR. AND GEST, H. 1954a Fuma-rate reduction by molecular hydrogen in cell-free systems. Bacteriol. Proc., 1954, 112.

PECK, H. D., JR. AND GEST, H. 1954b En-zymic reduction of pyridine nucleotides bymolecular hydrogen. Biochim. et Biophys.Acta, 15, 587-588.

SHUG, A. L., WILSON, P. W., GREEN, D. E., ANDMAHLER, H. R. 1954 The role of molyb-denum and flavin in hydrogenase. J. Am.Chem. Soc., 76, 3355-3356.

WHITELY, H. R. 1953 The mechanism of pro-pionic acid formation by succinate decarb-oxylation. I. The activation of succinate.Proc. Natl. Acad. Sci. U. S., 39, 772-779.

WILSON, J., KRAMPITZ, L. O., AND WERKMAN, C.H. 1948 Reversibility of a phosphoroclasticreaction. Biochem. J. (London), 42, 598-600.

WILSON, J. B. AND WILSON, P. W. 1942 Biotinas a growth factor for rhizobia. J. Bac-teriol., 43, 329-341.

WI¶"ER, L. D. 1953 Studies on the hydrogenaseactivity and on the metabolism of pyruvicacid in Micrococcus lactilyticus. Thesis,Univ. of Washington, Seattle, Washington.

WOLFE, R. S. AND O'KANE, D. J. 1953 Cofac-tors of the phosphoroclastic reaction ofClostridium butyricum. J. Biol. Chem., 205,755-765.

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