SYMPOSIUM ON METABOLISMI OF INORGANIC CO.'MPOUNDS'

III. NITROGEN FIXATION BY ENZYME PREPARATIONS

LEONARD E. MORTENSON,2 HOWARD F. MOWER, AND JAMES E. CARNAHAN

Central Research Department,3 Experimental Station, E. 1. do Pont de Nemours and Company,Wilmington, Delaware

I. Introduction ...........................................................................II. Extraction of Enzymes..................................................................

III. Requirements for Fixation ...........................................................IV. Formation of NH3 ........................................................

V. Components of Nitrogen-fixing System from Clostridiuim pasteurianumn...........VI. Roles of Fractions from Clostridium pasteurianutnm in Nitrogen Fixation ..................

A. Preparations from Cells Grown on NH3.............................................B. Effects of Carbon Monoxide............................................................

VII. Literature Cited......................................................................

1. INTRODUCTION

The atmosphere is the ultimate source ofnitrogen for all forms of life. However, atmos-pheric nitrogen is relatively inert and can bemetabolized by only limited numbers of special-ized organisms that are endowed with remarkablecatalytic powers over this element. The signifi-cance of biological nitrogen fixation to agricultureand also to chemistry has long been recognized,but in spite of much work the mechanism is stilla mystery. The obstacle has been the lack ofsuccess in extracting the responsible enzymes forstudy outside the cell (see, for example, Nason

and Takahashi (10)). This obstacle was recentlyremoved with the discovery of methods for pre-

paring active, cell-free extracts of the nitrogen-fixing systems from Clostridium pasteurianum(Carnahan et al. (3, 4)), blue-green algae (Schnei-der et al. (15)), Rhodospirillum rubrum (15),Azotobacter vinelandii (Nicholas and Fisher (12,13)), Chromatium (Arnon, Losada, and Nozaki(1)), and Bacillus polymyxa (Grau and Wilson(6)).This review summarizes progress made to date

I This symposium was organized by Dr. HowardGest under the auspices of the 1)ivision of GeneralBacteriology and presented at the Annual Meetingof the American Society for Microbiology inChicago, Ill., on April 26, 1961. Dr. Jacques C.Senez served as honorary convener.

2 Present address: Department of BiologicalSciences and/or School of Civil Engineering,Purdue University, Lafayette, Indiana.

3Contribution no. 696.

in understanding the chemistry of nitrogen fixa-tion by these enzyme preparations.

II. EXTRACTIONS OF ENZYMES

The nitrogen-fixing enzyme system has beenextracted from C. pasteurianum by two methods:(i) crushing frozen cells in a Hughes press, and(ii) autolysis of vacuum-dried cells (4). In theHughes press method, the frozen, crushed-cellmixture is thawed and centrifuged to obtain theactive, cell-free extract. In the dried-cells method,a cell paste from 10 liters of culture is driedrapidly without freezing in a rotary, vacuum

evaporator in a water bath at 30 to 40 C. Theresulting cell powder is extracted by mixing 10 g

with 100 ml of 0.05 M phosphate buffer, pH 6.8,and centrifuging at 30,000 X g for 15 min at 0 C.The dried cell extraction technique has been

used successfully also to obtain active, cell-freepreparations from R. rubrum (15). For disrup-tion of blue-green algae, the Raytheon 10 kcmagnetostrictive oscillator was used (15). Ex-tracts from A. vinelandii were prepared by a

modification of the lysozyme method of Repaskeand also by use of a Mullard 20 ke ultrasonicprobe (12, 13). In each case, nitrogen-fixing ac-

tivity was found in the supernatant solution aftercentrifugation at 144,000 X g for varying periodsup to 4 hr.

Ill. REQUIREMENTS FOR FIXATION

Fixation of -N2 by- these enzyme systems, ob-

tained free from cellular debris, is coupled withthe metabolism of various substances, not all of

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which are as yet identified. These auxiliary sub-strates, which clearly must serve as electrondonors, may serve other functions as well. Nosingle substrate has been found that will supportfixation in all of the solubilized systems; and, infact, a high degree of selectivity is evident at thepresent stage of progress.The C. pasteurianum enzyme preparations fix

nitrogen only when supplied with pyruvic ora-ketobutyric acids (4). Other compounds testedhave not replaced these substrates in initiatingor sustaining fixation by crude extracts. Theability of a-ketobutyrate to substitute for pyru-vate has been observed previously in other pyru-vate systems but with some exceptions, such asin the acetoin reaction (Dolin and Gunsalus (5)).The concentrated extracts appear to contain allrequired enzymes and cofactors, since additionof other factors has not proved stimulatory tofixation, with the exception that some extractsrespond to added coenzyme A. The specificity

of the pyruvate or a-ketobutyrate requirementmay reflect a dependency of fixation on somederivative of pyruvate metabolism in addition toelectrons, possibly an intermediate of the clos-tridial phosphoroclastic reaction sequence; but,if so, it remains to be identified. Nitrogen fixa-tion, even bv fresh extracts, does not begin untilafter a substantial quantity of pyruvate has beenconsumed (Fig. 1); and it continues in accom-paniment with pyruvate metabolism for periodsof 2 hrs or more with supplemental additions ofpyruvate at intervals of 10 to 15 min. Reducingagents, such as H2, sodium dithionite, and so-dium borohydride, and also various substrate-enzyme combinations that serve as reduced DPNregenerating systems have failed to replace pyru-vate in driving the fixation reactions.The enzyme preparations so far obtained from

R. rubrum require either pyruvate or a-keto-glutarate for nitrogen fixation (Burris and Wang(2)). Extracts of another photosynthetic or-

Inz0

0

0roE

0wxU.

zw

0

I-z

0

Ea

I00

(0

z0uw4

0

v 10 20 30 40 50 60-TIME (minutes)

FIG. 1. Time course of N2 fixation and pyruvate utilization by Clostridium pasteurianum extracts. Reac-tion mixture of 50 ml volume contained 0.05 M potassium phosphate buffer (pH 6.5) and 18 ml cell-free ex-tract containing 4 mg protein N per ml. Experiment was performed in a 250-ml flask containing 0.5 atm60 atom % N2"5 and 0.5 atm He. An alkali trap was attached to remove C02 generated by pyruvate metabo-lism. At the times indicated, 2.7 mmoles of sodium pyruvate were injected into the flask through a rubberadapter. Samples were removed at times indicated by points on curve and analyzed for pyruvate and atom% excess Nir.

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MORTENSON, MOWER, AND CARNAHAN

ganism, Chromatium sp., fix nitrogen when sup-

plied with hydrogen in the dark (1). Active prep-

arations of A. tinelandii are obtained only whenthe cells are disrupted in the medium in whichthey were grown, but the identity of the essentialfactor is not yet known (12, 13). Blue-green algalextracts have shown only weak activity, whichhas not been improved appreciably by additionof various compounds or by exposure to light(15).

IV. FORMATION OF NH3Mechanism studies on nitrogen fixation with

cell-free enzyme preparations show that NH3 isthe final product (4) as was previously suggestedfrom findings on intact cells (Zelitch et al. (18);Newton, Wilson, and Burris (11)). Thus far,discrete intermediates between N2 and NH3 havenot been demonstrated (4). Efforts to detecthydrazine, hydroxylamine, nitrite, and dihydro-pyridazinone-5-carboxylic acid as fixation inter-mediates in the C. pasteurianum system (4) todate have not been successful.

Tracer methods were employed in an examina-tion of the C. pasteurianum extracts for fixationproducts (4). Essentially all of the N21 taken up

by these preparations can be recovered as NIIH3by distillation from mildly alkaline solution.Conditions which permitted differentiation be-tween free NH3 and NH3 that was formed byamide hydrolysis during distillation showed over

90% of the freshly fixed N,"5 was present as freeN1H,3. The balance was distributed betweenamide and nonhydrolyzable, but Kjeldahl-di-gestible, derivatives.The crude extracts incorporate NH3 into or-

ganic derivatives in varying degree, and in some

instances this reaction has affected N' H3 re-

coveries. Thus, with extracts having high NH3-incorporating activity, the recovery of N1'H3 was

equivalent to N2" fixation during the first 10 to15 min and declined thereafter to about 10% bythe end of 1 hr. The N" in such preparations was

concentrated in glutamic acid. Prevention ofN"IH3 losses was accomplished by addition ofNH3 or glutamine before the start of fixation.This procedure permitted the recovery of at least90% of the N21, as N"5H3 throughout the periodof measurement, usually 1 hr (Table 1). FreeNH3 inhibited fixation only when its concentra-tion exceeded 10 to 12 ,umoles per ml. No in-hibition by glutamine was noted.

Since nitrogen fixation in the C. pasteurianum

TABLE 1. Influence of NH4+ and glutamine on NH4+accumulation during N2" fixation

N2 fixed

AdditionsaTotalb as NHic % as

NH3

Ag jAg

1. None.................. 244 10 42. NH4+, 120 umoles...... 214 182 903. Glutamine, 120 pmoles 240 250 104

aProtocol in a 25-ml flask: 400 pmoles phosphate(pH 6.5), 15 mg protein N as cell extract, 900pmoles sodium pyruvate (in side arm), in a totalvolume of 8 ml. The gas phase was 0.6 atm of 61atom % N2,". The pyruvate was tipped in and theflasks were incubated at 30 C for 60 min withshaking.

bTotal digestion by Kjeldahl method, followedby N15 analysis.

c Alkaline distillation (12).

preparations produces NH3 quantitatively, atitrimetric assay based on analysis for NH3 by anmodification of Conway's micro technique hasbeen developed to replace the laborious N'5method (Mortenson (8)). A radioactive tracerassay based on N213 also has been developed(Nicholas, Silvester, and Fowler (14)). It isextremely sensitive, but experiments must becarried through very rapidly because of the shorthalf-life (10.05 min) of this isotope.

V. COMPONENTS OF NITROGEN-FIXING SYSTEMFROM CLOSTRIDIUM PASTEURIANUM

Fractionation of the nitrogen-fixing extracts ofC. pasteurianum (Fig. 2) has resulted in theseparation of two fractions which individuallydo not fix nitrogen but which when combinedin appropriate proportions restore the full ac-tivity of the extract (Mortenson (7)). Althougha complete analysis of the roles of these twofractions has not yet been possible, there arestrong indications that the one designated NASin Fig. 2 contains the long postulated nitrogenaseenzyme. The other, designated HDS in Fig. 2,contains the clostridial phosphoroclastic systemas well as hydrogenase and is the source of re-ducing activity. Nitrogenase is assayed by fixa-tion activity in the reconstituted system and byspectral response to N2 (4). The clostridial phos-phoroclastic activity is assayed by pyruvateconsumption, acetyl phosphate production, and

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Vacuum-driedcells, 20g

Extract anaerobically for 60 min at 30 C in 200 ml 0.05 M potassium phosphatebuffer pH 6.5. Centrifuge at 0 C for 15 min at 35,000 X g.

Soluble fraction, "cell extract," fixes nitrogen (20 to 30 mg proteinper ml)

Heat at 60 C for 10 min underH2, cool to 0 C, centrifuge 10min at 35,000 X g and 0 C.

Residue

discard

Add protamine sulfate atratio of 1 part per 8 partsprotein at pH 6.0. Centrifugeat 0 C and 35,000 X g.

Soluble fraction 16 to 24 mgprotein per ml

Adjust pH to 6.5 andpotassium phosphateconcentration to 0.05M, add 150 mg calciumphosphate gel (dry wt)per 100 mg protein,mix anaerobically at0 C for 15 min, centri-fuge at 0 C for 5 minat 35,000 X g.

Soluble fraction 10 to 15mg protein per ml, re-ductase fraction or hy-drogen-donating system

HDS

Gel + adsorbed Supernatant solution 8 to 12material discard mg protein per ml, nitrogenase

fraction or nitrogen-activat-ing system

NAS

FIG. 2. Preparation of the nitrogen-activating (NAS) and hydrogen-donating (HDS) systems from ni-trogen-fixing extracts of Clostridium pasteurianum.

H2-CO2 evolution. Hydrogenase is assayed byhydrogen reduction of methylene blue.

Nitrogenase fractions, essentially free of phos-phoroclastic and hydrogenase activities, can beobtained by treating the C. pasteurianum extractswith protamine sulfate followed by calcium phos-phate gel at pH 6.5 (Fig. 2). These proceduresremove more than 80% of the nucleic acids and50% of the protein to leave a fraction, devoid ofnitrogen-fixing activity, containing 8 to 12 mg

protein per ml and less than 0.3 mg nucleic acidper ml (280:260 ratio of 1.2 to 1.5). On exposureto N2 this fraction usually shows an increasedabsorption in the vicinity of 300 mIA. This ab-sorption change is also characteristic of theoriginal extract (4) but cannot at present bedemonstrated in all fractions in proportion tonitrogenase activity. This fraction retains lessthan 5% of the pyruvate-metabolizing and hy-

drogenase activities of the orginal extract as

measured by evolution of CO2 and H2 and byreduction of methylene blue. Most preparations,however, are devoid of these activities when theirprotein content is no more than 49% of that inthe original extract. Separation of reducing ac-

tivities from this fraction prompted experimentsto prepare hydrogenase and pyruvate-metab-olizing fractions deficient in nitrogenase with theobjective of reconstituting fixation activity.

Purification of hydrogenase from C. pasteuri-anum was accomplished by Shug, Hamilton, andWilson (16) by heating cell extracts at 60 C for10 min under H2. This treatment was reportedto remove about 50% of the protein and virtu-ally all of the pyruvate-metabolizing activitywithout loss in hydrogenase activity. In our

laboratories, C. pasteurianum extracts (20 to 30mg protein per ml) heated at 60 C for 10 min

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TABLE 2. Nitrogen fixation by Clostridiumin pas-teurianum enzymes: determination of two

protein requirements

Additionsa Protein-N Nitrogen

mg lg N/16

1. Cell extract,b prepara-tion 1.................. 13.6 188

2. "Nitrogenase fraction"(NAS) ................. 9.1 0

3. Reductase fraction(HDS) ................. 6.8 0

4. Addition 2 + 3 ........ 9.1 + 6.8 1185. Cell extract, b prepara-

tion 2.18.6 2336. "Nitrogenase fraction". 14.8 07. Reductase fraction ..... 10.8 08. Addition 6 + 7 ........ 14.8 + 10.8 206

a Protocol otherwise as in Table 1.b Fractions as designated in Fig. 2.Samples 1 to 4 and 5 to 8 represent two separate

preparations of extract and fractions.

(Fig. 2) did indeed retain all the hydrogenaseactivity of the original extracts in solution; but,in contrast to the published results, more than95% of the pyruvate-metabolizing activity (meas-ured by reduction of methylene blue under argon)was present also. This 60 C heat-stable fractiondid not fix nitrogen.

Recombination of the two fractions just de-scribed, as outlined in Fig. 2, restored nitrogenfixation as shown in Table 2. Preparations of thenitrogenase fraction are stable for over 2 weeksif kept under H2 and at 0 C, whereas preparationsof the pyruvate-metabolizing fraction lose 50%or more of their activity on storage for 24 hrunder these same conditions.Some further insight into the soluble compo-

nents of the C. pasteurianum nitrogen-fixingsystem has been gained by dialysis of the cellextract and of the twvo components prepared fromit (NAS and HDS, Fig. 2). Dialysis of the cellextract for 24 hr at 5 C under H. or N2 resultedin loss of nitrogen-fixing activity. Dialysis of the60 C heat-treated fraction (HDS, Fig. 2) de-stroyed its ability for metabolizing pyruvateand for reactivating fixation when combinedwith the nitrogenase fraction (NAS, Fig. 2). Onthe other hand, dialysis of the nitrogenase frac-tion under these conditions does not remove itsability to fix nitrogen when recombined with the

60 C heat-stable fraction (HDS, Fig. 2). Further-more, nitrogen-fixation activity could be re-stored to dialyzed cell extracts by addition of the60 C heat-stable fraction (HDS, Fig. 2). Accord-ingly, nitrogenase in both the cell extract andin the protamine and phosphate gel fraction(NAS, Fig. 2) is stable to dialysis, whereas thepyruvate-metabolizing system is not.Treatment of the cell extract and the 60 C heat-

stable fraction (HDS, Fig. 2) with Dowex I resinat 0 C reduced the activity for fixing nitrogen inthe former and the activity for restoring nitrogenfixation to combinations with nitrogenase frac-tions in the latter. Both dialysis and Dowex 1treatment are presumed to remove essential co-factors. These have not been replaceable so farby addition of known cofactors for the clostridialphosphoroclastic reaction or by boiled cell ex-tracts (see Tables 3 and 4).

VI. ROLES OF FRACTIONS FROM CLOSTRIDI UMPASTE URIANUM IN NITROGEN FIXATION

Assignment of nitrogen absorption (nitrogen-ase) and hydrogen-donating functions, respec-tivelv, to the two required fractions (Fig. 2) pre-pared from N2-grown C. pasteurianum extractswas made initially on the basis of the spectralresponse to N2 found in the one and on the phos-phoroclastic and hydrogenase activities found inthe other (see Section V). This view of their

TABLE 3. Effect of dialysis and Dowex 1 treatmenton the activity of cell extracts and the reductasefraction (HDS) prepared from these extracts

Additionsa Pro- Nitrogentein-N fixed

nizg 51g N160mrgin

1. Cell extract.................... 15 2652. Cell extract dialyzed ......... 15 03. Cell extract dialyzed and

treated with Dowex lb ........ 15 04. 1 + reductasec. 7.8 2705. 2 + reductase.7.8 2586. 3 + reductase ................... 717. 1 + Dowex 1 treated reductaseb. 7.2 2608. 2 + Dowex 1 treated reductase. 7.2 1619. 3 + Dowex 1 treated reductase 7.2 12

a Protocol otherwise as in Table 1.b The preparation was mixed anaerobically with

Dowex 1 resin (Cl- form) at 0 C and the resinwas removed by centrifugation.

c HDS of Fig. 2.

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TABLE 4. Demonstration of heat lability of 60 C,10-min fraction and loss of a factor from

extracts by dialysis

Additionsa Pro- Nitrogentein-N fixed

mg gg N1601. Cell extract ............. 17 1652. Cell extract dialyzed.......... 15 83. 1 + reductaseb ........... 10 1754. 2 + reductase ............ 10 1585. 2 + reductase (85 C) c ......... 10 2306. 2 + reductase (95 C)d......... 5 1087. 2 + reductase (100 C)d ........ 2 0

8. "Nitrogenase"e ........... 10 89. 8 + reductaseb ........... 10 236

10. 8 + reductase (85 C)c......... 10 10011. 8 + reductase (95 C)>d......... 5 3012. 8 + reductase (100 C)d........ 2 0

a Protocol as in Fig. 2.b HDS fraction of Fig. 2.c Reductase was heated in water bath to the

temperature indicated and cooled immediately.d Equivalent volume to reductase in line 5.6 NAS fraction of Fig. 2.

functions has been supported by findings onextracts from NH3-grown cells and on inhibitoryeffects of carbon monoxide. To date, however,results do not reveal the nature of the reactionbetween nitrogen and nitrogenase, the first eventin fixation. Physical adsorption may be a possi-bility; but a reduction, oxidation, or additionalreaction might also be an integral part of theinteraction of the element and enzyme. Further-more, at the present stage, it is not certain thatnitrogenase is a single enzyme rather than oneof a series of enzymes acting to convert nitrogento ammonia in stepwise fashion.

A. Preparations from Cells Grown on NH3Cells of C. pasteurianum from NH3-grown cul-

tures do not yield nitrogen-fixing extracts whensubjected to the same procedures that give activeextracts from N2-grown cultures (7). The defecthas been traced to the nitrogenase fraction, whichwas inactive. Significantly, the pyruvate-metab-olizing fraction was present and functioningadequately. These conclusions are based on fixa-tion experiments in which the presence or absenceof activity in NAS and HDS fractions (Fig. 2)from NH3-grown cells was established by coupling

each with its complementary fraction from N2-grown cells (Table 5). The possibility of obtain-ing extracts of other organisms that will couplewith one or the other of these fractions to producenitrogen-fixing activity is being investigated.The observation has often been reported that

nitrogen-fixing organisms do not fix nitrogenwhen NH3 is available. Thus, fixation activityappears to be an inducible (or repressible) char-acter. The experiments described in Table 5suggest that the inducible (or repressible) com-ponent in nitrogen fixation is in the NAS fraction(Fig. 2) of the extracts from C. pasteurianum.

B. Effects of Carbon Monoxide

Carbon monoxide inhibits nitrogen fixation bycell-free preparations from C. pasteurianum. Itappears to react with at least three componentsof the extracts, namely, hydrogenase and twofixation mediators as described in followingparagraphs (Mower (9)). One of the mediatorsis in the nitrogenase fraction, and the other isin the pyruvate-metabolizing fraction along withhydrogenase. They can be differentiated on the

TABLE 5. Fixation activity in combined fractionsfrom N2-grown and NH3-grown

Clostridium pasteurianum

Additionsa Pro- Nitrogentein-N fixed

NH3-Nmg formed/

60 mni1. Cell extract N2-grown cells.... 15 3012. Cell extract, NH4+-grown cells. 15 03. "Nitrogenase" fraction,b N2-

grown cells ................... 6.9 04. "Nitrogenase" fraction,b NH4-

grown cells ................... 7.5 05. "Reductase fraction," N2-

grown cells ............ ....... 8.1 06. "Reductase fraction," NH4+-

grown cells ................... 9.3 07. 3 + 5 ... 2908. 3 + 6 ... 157c9. 4+5 ... 010. 4+ 6 ... 0

a Protocol as in footnote, Table 2.b Nitrogenase (NAS) and reductase (HDS) frac-

tions were prepared from N2- and NH4+-growncells as described in Fig. 2.

c Pyruvate metabolized by this fraction atabout 50% the rate of the similar preparationfrom N2-grown cells.

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Ow

D0LU

z0

(L)

w

a.

0E

5000 F

4000

3000F

2000

1000

0

I -I* ' -

"a

PYRUVATE a

A--0

o o

/ 0~~~~~~~~0a~~~~~~~aD/0

NN FIX(I. I 44

10 20 30 40

TIME (min.)

50 60

20 'en4z

150z

10 aw

5Z4iE~L

-770

FIG. 3. Effect of CO exposure to NAS and HDS in recombination experiments. 0 = control, No COtreatment of either NAS or HDS (see Fig. 2); A = HDS, exposed to CO 15 min, pumped off, NAS added,and El = NAS, exposed to CO 45 min, pumped off, HDS added.

Right ordinate: Micrograms of nitrogen fixed per mg protein nitrogen in nitrogen-activating fraction.Each flask contained 5 mg protein nitrogen as hydrogen-donating fraction, 5 mg protein nitrogen as nitro-gen-activating fraction, 80 mg Na-pyruvate, and 100 Mmoles phosphate. Total volume of 3.0 ml, pH 6.8.

basis of the relative rates of formation and dis-sociation of their carbon monoxide complexes.Time-course data on nitrogen fixation with prep-

arations in which the two sensitive fixation medi-ators were inhibited one at a time are in accordwith the view that the fraction designated NAS(Fig. 2) contains nitrogenase, and the fractiondesignated HDS (Fig. 2) produces electrons andany other substrate that may be required frompyruvate.

Selective inhibition of these components was

accomplished by exposing the two enzyme frac-tions to carbon monoxide separately. Exposureswere for varying times, after which the carbonmonoxide atmosphere was replaced by H2 or N2.Fixation activity was then measured by com-

bining the carbon monoxide-treated fraction withits untreated complementary fraction, addingsodium pyruvate, and exposing to N2. Resultsof such experiments are shown in Fig. 3.The effect of carbon monoxide on the pyruvate-

metabolizing fraction was to prolong the induc-tion period before nitrogen fixation started; afterthis temporary inhibition, nitrogen fixation pro-ceeded at the rate observed in untreated controls.The maximal induction period was produced by

15 min of exposure to carbon monoxide; afterremoval of carbon monoxide, its effect was re-

lieved within 15 min under N2. The rate ofpyruvate metabolism measured by methyleneublue redction proceeded in the presence of car-

bon monoxide at 90 to 95% of the rate in theabsence of carbon monoxide. Consequently, theblock would appear to be in the electron transportsteps toward nitrogen fixation or on an aspect ofpyruvate metabolism not involved in H2 pro-

duction.The effect of carbon monoxide on the nitro-

genase fraction was to decrease the rate of nitro-gen fixation without affecting the length of theinduction period (Fig. 3). The degree of inhibitionincreased progressively with increasing exposures

to carbon monoxide and was not complete untilafter about 16 hr (Fig. 4). Pyruvate metabolismas measured by methylene blue reduction was notaffected. Formation of this carbon monoxidecomplex proceeded relatively slowly and was notreversed by evacuation to remove carbon mon-

oxide as shown by nitrogen-fixation measure-

ments. The block therefore appeared to involvea nondialyzable component (see Section V) whichfunctions in the rate-limiting step of nitrogen

o ,/7'11

ATION

l

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90

70z

0

° 60 _

'50-z

S 40

30

20

I0

0 5 10 15 20 25 30 35 40

TIME OF EXPOSURE (min.)

FIG. 4. Inhibition of fixation by exposure of NASfraction to CO for varying lengths of time. Afterexposure, the CO atmosphere was replaced by hydro-gen until the fraction was assayed for nitrogen-fixingactivity.

fixation but not in the pyruvate-metabolizingsystem.

In the course of these experiments, the in-hibition of hydrogenase by carbon monoxide(Shug and Wilson (17)) was confirmed. Reductionof methylene blue by H2 in the C. pasteurianumextracts was blocked completely by carbon mon-

oxide. This inhibition was not time dependentbut occurred immediately on addition of carbonmonoxide and was relieved immediately whenthe atmosphere of carbon monoxide was replacedwith hydrogen. Reduction of methylene blue bysodium pyruvate in the extracts was not sensitiveto carbon monoxide. The route of elecrtons frompyruvate to methylene blue must differ fromthat to nitrogen because carbon monoxide doesnot affect the former but does affect the latter.The initiation and release of carbon monoxideinhibition of hydrogenase was too rapid to per-

mit tests on the possible involvement of hy-drogenase in nitrogen fixation by this type ofexperiment. However, nitrogen fixation has beenreported to occur in A. tinelandii extracts afterremoval of hydrogenase by centrifugation (14).The hydrogenase was assayed by methylene bluereduction. This observation does not eliminatethe possibility of common intermediates in elec-tron transport between pyruvate and H2 andbetween pyruvate and N2 that may act reversibly

and permit nitrogen reduction by hydrogen underappropriate conditions (1). So far, however, hy-drogen has been inactive, and pyruvate (or a-ketobutyrate) remains an essential substrate fornitrogen fixation in the C. pasteurianum system.

VII. LITERATURE CITED1. ARNON, D. I., M. LOSADA, M. NOZAKI, AND

K. TAGAWA. 1960. Photofixation of nitrogenand photoproduction of hydrogen by thio-sulfate during bacterial photosynthesis.Biochem. J. 77:23P-24P.

2. BURRIS, R. H., AND L. C. WANG. 1960. Fixationof nitrogen by cell-free preparations fromRhodospirillum rubrum. Plant Physiol. 35:(Suppl.), xi.

3. CARNAHAN, J. E., L. E. MORTENSON, H. F.MOWER, AND J. E. CASTLE. 1960. Nitrogenfixation in cell-free extracts of Clostridiumpasteurianum. Biochim. et Biophys. Acta38:188-189.

4. CARNAHAN, J. E., L. E. MORTENSON, H. F.MOWER, AND J. E. CASTLE. 1960. Nitrogenfixation in cell-free extracts of Clostridiumpasteurianum. Biochim. et Biophys. Acta44:520-535.

5. DOLIN, M. I., AND I. C. GUNSALUS. 1952. Asoluble pyruvate ai-ketobutyrate dehydro-genase system from Streptococcus faecalis.Federation Proc. 11:203.

6. GRAU, F. H., AND P. W. WILSON. 1961. Cell-freenitrogen fixation by Bacillus polymyxa.Bacteriol Proc. p. 193.

7. MORTENSON, L. E. 1961. Components of thenitrogen fixation enzyme system in Clostrid-ium pasteurianum. Federation Proc. 20:234(abstract).

8. MORTENSON, L. E. 1961. A simple method formeasuring nitrogen fixation by cell-free en-zyme preparations of Clostridium pasteuri-anum. Anal. Biochem 2:216-220.

9. MOWER, H. F. 1961. Carbon monoxide inhibi-tion of enzymic N2-fixation. FederationProc. 20 349. (abstract)

10. NASON, A., AND H. TAKAHASHI. 1958. Inor-ganic nitrogen metabolism. Ann. Rev. Mi-crobiol. 12:203-246.

11. NEWTON, J. W., P. W. WILSON, AND R. H.BURRIS. 1953. Direct demonstration of am-monia as an intermedate in nitrogen fixationby Azotobacter. J. Biol. Chem. 204:445-451.

12. NICHOLAS, D. J. D., AND D. J. FISHER. 1960.Nitrogen fixation in extracts of Azotobactervinelandii. Nature 186:735-736.

13. NICHOLAS, D. J. D., AND D. J. FISHER. 1960.Nitrogen fixation in extracts of Azotobactervinelandii. J. Sci. Food Agr. 11:603-608.

1

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14. NICHOLAS, D. J. D., 1). J. SILVESTER) AND J. F.FOWLER. 1961. Use of radioactive nitrogen in

studying nitrogen fixation in bacterial cells

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