BtoCHIMICA ET BIOPHYSICA ACTA

BBA 6S2IO

THE ADENOSINE TRIPHOSPHATE REQUIREMENT FOR NITROGEN FIX­

ATION IN CELL-FREE EXTRACTS OF CLOSTRIDIUM PASTEURIANUM

M.]. DILWORTH*, D. SUBRAMANIAN", T. O. MUNSON AND R. H. BURlUSDepartment of Biochemistry, University of Wisconsin, Madison, Wise. (U.S.A.j

(Received December I rth, 1964)

SUMMARY

I. The requirements for the decomposition of acetyl phosphate in extracts ofClostridium pasteurianum have been shown to be the same as those for N2 fixationwith H2 as the electron donor-s-Hg, ATP, ferredoxin and Mg2+. The decompositionof acetyl phosphate (ATP) under H 2 is not caused by an activation of enzymes byH2, nor by an ATP-dependent uptake of substrate amounts of H 2•

2. Removal of ATP from extracts by charcoal treatment allowed N2 fixationfrom pyruvate to proceed appreciably when fixation from H 2 was almost completelyinhibited. Addition of increasing amounts of ATP to extracts freed of ATP bySephadex gel-filtration showed that a much lower concentration of ATP-supportedN2 fixation from pyruvate than from H 2• One site of ATP action was thus localizedbetween H2 and reduced ferredoxin.

3. A requirement for ATP in pyruvate metabolism has been established andmay explain the ATP requirement for N2 fixation from pyruvate. It appears doubtfulthat ATP is required at the site of N2 reduction.

4. H2 acted as a competitor of N2 fixation when H 2 was the electron donor,but acetyl phosphate (ATP) consumption increased with increasing pH2. When N2

fixation was completely inhibited by either CO or N20, ATP consumption was onlyslightly decreased. Separate enzymes apparently function in ATP consumption andN2 reduction.

5. Mg2+ is required for ATP consumption as well as for ATP generation fromacetyl phosphate by acetokinase (ATP:acetate phosphotransferase, EC 2.7.2.1).

6. A scheme is presented for the action of ATP in N2 fixation; an activatedform of reduced ferredoxin produced enzymically with ATP is considered to functionin N2 fixation.

'Present address: Dept. of Soil Science and Plant Nutrition, University of WesternAustralia, Perth, Australia.

" Present address: University Botany Dept., Madras 5, India.

Biocbim, Biophys. Acta, 99 (1965) 486-503

ATP AND N2 FIXATION

INTRODUCTION

The involvement of high-energy phosphate in biological N2 fixation was suggestedby McNARY AND BURRIS1, based on the finding that either arsenate or glucose-hexoki­nase (ATP: D-glucose 6-phosphotransferase, EC z.7.1.:+) strongly inhibited N2 fixationin extracts of Clostridium pasteuriamtm using pyruvate as the electron donor. It hasbeen suggested- that the inhibition by glucose was at least partly due to phosphatedepletion, and the further possibility of inhibition by glucose o-phosphate formed dur­ing the reaction must be considered. However, the inhibition by arsenate, whichprevents ATP synthesis from acetyl-CoA via acetyl phosphate, was very significant.

Requirements for ATP during fixation of N2 with electrons from pyruvate,NADH, borohydride" and hydrogen gas3,4 have been shown for the system fromC. pasteurianum, and for particles from Azotobacter vinelandii5 using H2. Tentativemechanisms advanced to account for the ATP involvement include formation of anactivated oxidized nitrogenase enzyme", or an activated state of the reduced nitro­genase-, Both these mechanisms imply one site of action for ATP and that will be thesite of N2fixation. However, no conclusive evidence for the involvement of ATP spe­cifically at the site of N2 reduction has yet been reported. In this paper evidence ispresented suggesting that there are two sites for ATP action, and that one of thesemay be concerned with electron transport between H2 and ferredoxin.

METHODS

BacteriologicalCultures of C. pasteurianum strain W5 were grown with N2 in a nitrogen­

deficient medium" with the final stage in a 150-1 glass-lined fermentor maintained at30° and at pH 6.5. Cultures grown with ammonium ion as nitrogen source containedan additional 750 mg (NH4)2S04 per 1 and were swept slowly with argon. Cells wereharvested according to DUA AND BURRIS?, dried at 40-45° under vacuums, and storedat room temperature in vacuo in sealed ampoules. Such storage allowed the prepara­tion of extracts with reproducible activities for at least 9 months.

ReagentsReagents were purchased as follows: CoA, ATP and sodium pyruvate from

Sigma Chemical Co., DEAE-cellulose (0.89 mequivjg) from Schleicher and Schuell Co.and Sephadex G-z5 from Pharrnacia, Sweden.

Dilithium acetyl phosphate was synthesized from acetic anhydride": it assayedat 72 % purity based on a succinic anhydride standard.

Charcoal (Darco G-60, Atlas Powder Co.) was refluxed for 6 h in 6 N HCl andwashed free of acid before drying at r ro".

Gas mixtures were prepared and stored over saturated NaCl solution and dis­placed into the flasks with it. H2 was passed through a Deoxo catalytic purifier(Engelhard Industries).

Ferredoxin was isolated and crystallized from C. pasteuriamtm as describedby MORTENSON10, and concentrations were determined spectrophotometrically fromthe molar absorbancy of 33.2 at 390 mf-t (ref. 10).

Biochim: Biopbys. Acta, 99 (1965) 486-503

M. J. DILWORTH et at.

Preparation and treatment of extractsExtracts were prepared by autolysis of the dried cells in 50 mM buffer (phos­

phate or cacodylate) at pH 6.7. or in water, by shaking under H 2 for I h at 32°.The crude extract obtained after centrifugation (25000 X g, IS min) at 4° was useddirectly after gassing with H 2 and contained 10-15 mg protein per ml.

ATP was removed from the crude extract by treatment with 5 or 10 mg ofacid-washed charcoal per ml of extract. The charcoal was washed once with 50 mMpotassium phosphate buffer (pH 6.7), centrifuged down and transferred to a suctionflask. After flushing the flask with H 2, crude extract was added under a stream of H 2 ,

the flask was filled with H 2 and the extract stirred for IS min at IS° before centrifuga­tion (8000 X g, 5 min) to remove the charcoal. Treatment with larger amounts ofcharcoal (z5-30 mg per ml of extract) removed appreciable amounts of ferredoxinas well as ATP.

ATP and Mg2+ were also removed by passing the extract through an anaerobiccolumn (z3 em X 2 em diam.) of Sephadex G-25 in 50 mM phosphate buffer (pH 6.7)at a flow rate of 0.7 mljrnin in an apparatus similar to that designed by SAKAMIll .

Bivalent metal ions were removed by treatment with Chelex roo (Calbiochem,100-ZOO mesh, Na.! form) at a rate of I g wet resin per 190 mg protein; handlingtechniques were similar to those used for charcoal treatment except that the extractwas sucked off through a cotton-plugged pipette.

Ferredoxin was removed with DEAE-cellulose as described by MORTENSON12•

One extract from each batch of cells was titrated before use; amounts of DEAE­cellulose from 0.75 to 3.0 g per g of extract protein were required to prevent N2 fixationand acetyl phosphate decomposition.

AnalyticalN2 fixation was measured by NHs formation in zo-ml rubber-capped serum

bottles filled with the desired gas through a No. 18 hypodermic needle by repeatedevacuation and filling. The bottles were shaken at 250 strokes per min in a water bathat 32°. Reaction was initiated by the addition of extract by hypodermic needle throughthe rubber cap and terminated at 30 min by the addition of I ml of saturated K2COS

solution after removal of samples for acetyl phosphate determination. Ammonia wasrecovered by diffusion into I N H 2S0 4 carried on the end of a glass rod supported inthe serum bottle by a rubber stopper; the bottles were rotated mechanically forz h, the glass rod then dipped into Nessler's reagent"; and the color estimated.

When H 2 was the electron donor, the standard incubation mixture contained25 ,umoles potassium phosphate buffer (pH 6.7),2.5 ,umoles Na2ATP, 5 ,umoles MgCl2 ,

50 ,umoles dilithium acetyl phosphate and 3-5 mg protein from C. pasteurianum in a:finalvolume of I.O mI with a gas space Oi20 mI. When pyruvate was the electron donor,the reaction mixture contained 15,umolespotassium phosphate buffer (pH 6.7), 50 ,umo­les sodium cacodylate buffer (pH 6.7), 50-ISO,umolesofsodium pyruvate (optimum de­termined by titration with each batch of cells), 5 ,umolesMgC12, 2.5,umolesNa2ATP and3-5 mg protein from C.pasteurianum in a final volume of 1.0 ml with a gas space of 20ml.

Acetyl phosphate was assayed as acetohydroxamic add14 , and pyruvate as thez,4-dinitrophenylhydrazone15• Acetyl phosphate decomposition during fixation of N2

with H 2 was calculated from the difference between that in bottles under N2/H2 andthat in bottles under argon.

Biockim, Biopbys, Acta, 99 (1965) 486-503

ATP AND N2 FIXATION

Acetokinase (ATP:acetate phosphotransferase, EC 2.7.2.1) was assayed byhydroxamic acid formation from acetate and ATP in the presence of hydroxylamineie:in some assays the Tris buffer was replaced by cacodylate buffer (pH 6.7) to giveconditions more comparable to those used for N2 fixation.

Protein was measured by the biuret method with bovine serum albumin as astandard. Gas uptakes were measured by conventional techniques'" at 32° in thepresence of alkaline pyrogallol (0.08 rnl ro N KOH and 0.04 ml25 % (wjv) pyrogallol).

RESULTS

Assay conditionsOptimum conditions for assay of N2 fixation with H 2 as electron donor have

been studied, and the results are summarized in Figs. 1-6.The time course of N2 fixation with H 2 as electron donor and acetyl phosphate

as ATP generator is shown in Fig. I with data for acetyl phosphate decomposition.

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Fig. 1. Time course of N. fixation and acetyl phosphate decomposition. Reaction vessels (7o-mlWarburg flasks with a side port closed with a serum stopper) contained, in a final volume of4 ml, 70 ftmoles potassium phosphate (pH 6.7), 20,umoles neutral ADP, 2oltmoles MgCI.,148,umoles acetyl phosphate, and 12.6 mg protein, with alkaline pyrogallol in the center well.Extract was added, and samples withdrawn by syringe through the serum stopper. Gas phase:0.5 atm H. with 0.5 atm N. or 0,5 atm A. Acetyl phosphate decomposition: .-e, N./H.: 0-0,A/H•. N. fixation, .-•.

In agreement with MORTENSON4 , the presence of H 2 with either argon or N2 causesacetyl phosphate disappearance at a rapid rate, the early high rate occurring whileADP is first phosphorylated. In our initial experiments with MgS0 4 supplying Mg2+it appeared possible that H 2 could be used for sulphate reduction, a process known torequire ATP. However, identical results were obtained with MgCI2•

Biochim. Biopbys. Acta, 99 (1965) 486-503

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Fig. 2. Protein concentration and H,-dependent N. fixation and acetyl phosphate breakdown.Standard assay system. Gas phase: 0.5 atm H. + 0.5 atm N., or A. 0-0. acetyl phosphatedecomposition; e-e, N, fixation.

Titration of protein concentration showed that fixation and acetyl phosphatebreakdown were linear above I mg/ml (Fig. 2). For a 30-min assay period. approx.4 mg protein have been used. The lag in the curve may represent the amount of en­zyme needed to remove an inhibitor at the start of the reaction, possibly 02'

Acetyl phosphate is strongly inhibitory both to ATP breakdown and N 2

fixation; a concentration of 40 mM gave good fixation over the assay period used(Table I).

The optimal concentration of ADP or ATP for acetyl phosphate breakdownwas about 2 mM, and that for N2 fixation between 2 and 5 mM (Fig. 3). The break­down of acetyl phosphate under argon is usually slight (less than ro% of the acetylphosphate added) even in the presence of ATP; in this experiment it could be ac­counted for in terms of the phosphorylation of ADP.

The inhibitory effects of high concentrations of ATP have been noted previous-

TABLE I

EFFECT OF ACETYL PHOSPIJATE CONCENTRATION ON N. FIXATION AND ACETYL PHOSPHATE BREAK­

DOWN

Standard assay system. Gas phase: 0.5 atm N. + 0.5 atm H2• or 1 atm A. Protein: 4.3 mg.

Initial acetyl N 2!ixed Acetyl phosphatephosphate [mumoles /mg decomposedconcentration protein/min) (mftmoles/mg(mM) protein[mi«}

44 8.2 16989 3.8 II]

133 1.2 61178 0.3 0

Biochim. BioPhys. Acta. 99 (1965) 486-503

ATP AND N2 FIXATION

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Fig. 3. Effect of ATP concentration on N. fixation and acetyl phosphate breakdown. Standardassay system, with 3. I mg protein from a charcoal-treated extract. Gas phase: 0.,') atm II. +0.5 atm N., or A. 0-0, acetyl phosphate decomposition; .-e, N. fixation.

ly8 for clostridial extracts using pyruvate, and also for extracts using hydrogens,It is for this reason that a system generating a steady low concentration of ATP hasbeen found necessary. In contrast to other workers, we have shown NBs formationfrom H 2 with ATP alone; addition of 5 mM ATP to an extract and incubation foronly 5 min allowed fixation <'JJi 3.8 mzzmoles N2 per mg protein per min.

Full activity for both N2 fixation and ATP degradation was restored when5 roM MgH was added to extracts treated with Chelex 100 (Table II).

TABLE II

REQUIREMENTS FOR N. FIXAnON AND ACETYL PHOSPHATE DEGRADATION BY EXTRACTS OF C.pasteurianum. UNDER N./H. MIXTURES

Extract protein added: Expt. 1, untreated, 4.3 mg: treated, 4.0 mg; Expt. 2, untreated 4.0 mg;treated, 3.2 mg; Expt. 3, untreated, 3.6 mg; treated, 3.6 mg. The standard incubation mixturewas used in all experiments with omission of ATP in Expt, I and of MgCl. in Expt. 3. Gas phase:0.5 atm N 2 + 0.5 atm H., with controls under A.

E;rpt. Extract treatment Addition N • .fixed Acetyl phosphate(p,moles) (mJ.l.moles/mg used

Material mg adsorbent/ protein/min) (mp,moles/mgmg protein protein/min)

Charcoal 0·7 None 0.0 0Charcoal 0·7 ATP (5) 7. 1 157None ATP (s) 7. 6 145

DEAE-cellulose 3.0 None 0.0 32 D EAE-cellulose 3.0 Ferredoxin (0.018) 6.6 167

None None 9·7 190

Chelex roo lIO None 0.0 03 Chelex 100 lID MgCl. (s) 9·9 179

None MgCI. (5) 10·3 213

Biochim. Biophys, Acta, 99 (1965) 486~s03

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The initial report of N2 fixation in C. pasteurianan: extracts with pyruvate­indicated a narrow pH optimum between pH 6.3 and 6.5. With H2 the optimum ismuch broader, between pH 6.7 and 7.7. Fig . 4 is a composite curve obtained withTris-Hf.l and cacodylate buffers; to obtain a complete curve the values for Tris have

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Biochim , Biophys. Acta, 99 (1965) 486-503

ATP AND N2 FIXATION 493

been multiplied by a ratio between activities in both buffers over the range of over­lapping pH. Tris buffer of the same molarity and pH gave about five-sixths of theactivity found with cacodylate.

The apparent K m for H 2 was calculated to be 0.10 atm H2 for N2 fixationand 0.045 atm H 2 for acetyl phosphate (ATP) breakdown (Fig. 5). These are bothlower than the valu e of 0.28 atm previously reported for N2 fixati ons.

The apparent K m for N2 was determined at two different H 2 pressures, becauseH 2 inhibits N2 fixation by the same system using pyruvate as the electron donor-",At 0.1 atm H2 the apparent K m for N 2 was 0.05 atm ; at 0.5 atm H2 , 0.09 atm (com­pare K m for N2 of 0.17 atm with pH2 = 0.5 atm (ref. 4)). A K m of 0 .08 atm N2 wasestimated by SCHNEIDER18 for extracts of C. pasteurianum with pyruvate as electrondonor, and values of 0.03 (ref. 6) and 0.076 atm N 2 (ref. 18) have been reported foractively growing intact cells. From Fig. 6 it can be seen that H2 and N 2 are apparently

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competitive, in agreement with the results with the pyruvate system'". From the twocurves, K, was calculated as 0.32 atm, leading to a K m for N2 of 0.037 atm at zeropH2. Most of our experiments have been done using a mixture of 0.5 atm N2 and 0.5atm H2. It is interesting to note that increasing the pH2 from 0.1 atm to O.S atmincreased acetyl phosphate (ATP) breakdown while N2 fixation was inhibited.

One possible limitation of the assay lay in the dependence on acetokinase forATP generation. A rate of phosphorylation of acetate of 20 ,umolesperh per mg proteinhas been reported by MORTENSON2, and this would be compatible with the rate re­quired in these experiments (about 25 ,umoles per h per mg protein) as acetokinaseis known to catalyze acetyl phosphate conversion to ATP and acetate much fasterthan acetyl phosphate synthesis. Direct assays in this lab oratory place the figure ashigh as 360 ~moles acetyl phosphate per h per mg protein (assayed according toROSE10). so that acetokinase is not the limiting enzyme in the system .

The efficiency of N 2 fixati on in terms of ATP consumption is poor; the bestpreparations consumed 8.2 ,umoles acetyl phosphate (ATP) per ~mole NHs produced,or about 3 ATP molecules per electron transported to N 2•

Biochim, B iophy s, A cta, 99 (r965) 486-503

494 M. J. DILWORTH et al,

Possible mechanism for H 2-dependent acetyl phosphate disappearanceThree types of mechanism seemed possible for H 2-dependent acetyl phosphate

decomposition: (1) degradation of acetyl phosphate (ATP) coupled with substratelevel H 2 uptake into some endogenous acceptor, (2) H 2 activation of some enzyme(s)concerned in acetyl phosphate or ATP hydrolysis, or (3) a cyclic electron flow drivenin some way by ATP, with or without appreciable H 2 uptake.

In Fig. 7 the rates of gas uptake are shown for extracts metabolizing acetylphosphate under either N2/H2 or A/H2 mixtures. Clearly. there is no appreciableuptake of H2 under A/H2, and that under N2/H2 is exactly attributable to N2 fixation,again in agreement with MORTENSON4• Thus, explanation 1 cannot be true.

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Fig. 7. Gas uptakes and N. fixation. Reaction vessels (ao-ml Warburg flasks with alkaline pyro­gallol) contained: main compartment, r l.3 mg protein and 35 I~moles potassium phosphate(pH 6.7) in I.5 ml; side arm, 89 pmoles acetyl phosphate, 20 I~moles sodium cacodylate (pH 6.7),zo zzmoles MgCI. and ro jzmoles ATP in 0.5 ml, Gas phase: 0.5 atm H., with either 0.5 atm N.or 0.5 atm A; or 1.0 atm A. Gas uptake: .-e, N./H.; 0-0, A/H.; .-., difference; 0-0,calculated from NH. produced.

To test for an activation of hydrolytic enzymes by H 2 , crude extracts Wereallowed to use acetyl phosphate under either argon or H 2 atmospheres for IS min inthe presence of alkaline pyrogallol. At that time half the H 2 flasks were refilled withargon, the other half continuing under H 2 • while all received an injection of pre-gassedacetyl phosphate solution. From Fig. 8 it is clear that those flasks changed to argonassumed the rate characteristic of argon controls, while acetyl phosphate decomposi­tion continued unchecked in the H 2 series. It therefore appears that activation by H 2of enzymes concerned in acetyl phosphate breakdown (acetyl phosphate phospho­hydrolase, EC 3.6.I.7; ATP phosphohydrolase, EC 3.6.I.4, or ATP pyrophospho­hydrolase, EC 3.6.r.8) is not a satisfactory explanation. It remains to explain abreakdown of acetyl phosphate which is dependent on ATP, Mg2+ , H 2 and ferredoxin(Table II) and which does not involve the uptake of substrate quantities of H 2•

BiocMm. Biophys, Acta, 99 (1965) 486-503

ATP AND N2 FIXATION

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Fig. 8. Test for activation by H. of acetyl phosphate decomposition. Reaction vessels (7o-mlWarburg flasks as for Fig. r) initially contained 65/lmoles potassium phosphate (pH 6.7).+2 umoles acetyl phosphate, 30 pIDolcs MgCI. and IS Ilmoles ATP in a volume of 3.0 ml. At IS min(t) a c.y-ml sample was withdrawn from each flask: two flasks of the H. set were regassed withA. Each flask received a further 6o/lmoles acetyl phosphate in 0.4 ml, after which acetyl phos­phate breakdown was followed . .-e. H.; 0-0, A; .A.-•. A after He-

Fig. g. Effect of ferredoxin on N. fixation and acetyl phosphate breakdown. Standard assaysystem. with 3.2 mg protein from an extract treated with 3 g DEAE-eellulose per g protein.Gas phase: 0.5 atrn N. + 0.5 atm H., or A. 0-0, acetyl phosphate breakdown; .-e. Ntfixation.

Cofactor removalFerredoxin, the iron-sulphide protein concerned in electron transport in many

anaerobic bacteria, is essential for N2 fixation in C. pasteurianum extracts using eitherpyruvate or H 2 as electron donor 12,19. It is also essential for the decomposition ofacetyl phosphate in the presence of ATP and H 2 (Table II). By removal of ferredoxinwith DEAE-cellulose and addition of known quantities of ferredoxin to the extracts.it was possible to show that the half-maximum rate of ATP degradation occurredwith 9 flg ferredoxin per ml under 0.5 atm H 2 in N'2 or A (Fig. 9). The apparent K mfor N2 fixation was 13 f/;gjml. Since ferredoxin is the intersection point for electronsfrom pyruvate or H 2 and is essential for ATP hydrolysis by H 2• the site(s) of actionof ATP in N2 fixation can be localized by studying the effect of ATP depletion andrestoration on electron transfer from pyruvate or H 2o

Treatment with charcoal removes ATP from the extracts; activity can be al­most fully restored by the addition of ATP alone (Table III). As can be seen. theendogenous ATP sufficed for 62% of full activity with pyruvate and 23% with H 2·

Charcoal treatment reduced the fixation to rather less than half the endogenouslevel with pyruvate and almost completely inhibited fixation with H 2• In another

Biochim, Biopbys. Acta, 99 (1965) 486-503

M. J. DILWORTH et at.

TABLE III

THE EFFECT OF CHARCOAL TREATMENT ON N 2 FIXATION AND ACETYL PHOSPHATE DEGRADATIONBY EXTRACTS OF C. pasteurianwm.

Extract (ro ml, 146 mg protein) treated with JOO mg charcoal. Assayed with the standard assaymixtures; ATP omitted from both series; cacodylate omitted and o.oy umole CoASH added tothe pyruvate series. Gas phase: N 2 or A in the pyruvate series; 0.5 atm N, + 0.5 atm H, or Ain the H, series.

Addition Treatment of Substrate(remoles) extract

Pyruvate H,

(mremoles{ % N,fixed % Acetyl phos- %mg proteinl (mpmolesl phate usedmin) mg protein/ ( mumoles[mg

min) protein/min)

None Not treated 8·3 62 2.1 23 53 26ATP (2.5) Not treated 13·5 100 9. 2 100 206 100None Charcoal 3·3 25 o.r I II 5ATP (2.5) Charcoal 12.4- 92 8·3 9° 189 92

experiment, in which the endogenous ATP supported 60% of the maximum rate offixation with added ATP, treatment with 5 mg charcoal per ml extract had no effecton fixation from pyruvate but reduced that with H 2 to I3% of the control. Thatcharcoal treatment does not inactivate the system is apparent from the 90-92 %recovery of activity on addition of ATP. These experiments indicated a differentialeffect of ATP on H 2- and pyruvate-supported systems, and this in turn suggestedthat there was a requirement for ATP prior to ferredoxin in the H 2 pathway.

Certain difficulties arise in interpreting these experiments. The conditionsrequired for N 2 fixation from H 2 (Mg2+, and acetyl phosphate as a source of ATP)are met during pyruvate metabolisms, and it is therefore possible that N2 fixationcould proceed simultaneously from both H 2 and pyruvate as electron donors. Theapparent inhibition of N2 fixation from pyruvate by charcoal treatment might thenbe due to prevention of this "extra" fixation from H 2 evolved from pyruvate. It issuggestive that a 34% inhibition of N 2 fixation with pyruvate can be achieved byrapidly sparging high-purity N 2 through a fixing system, and that this inhibitionapparently is not attributable to enzyme inactivation. With the low apparent K m forN 2 fixation (Fig. 5), it is difficult to remove H 2 from solution to a point where in­hibition from deficiency of H 2 as an electron donor becomes detectable. Despite thedifficulty, inhibition by sparging was clearly established.

Activities for N2 fixation with H 2 or pyruvate appear to bear a constant ratioto one another when both are maximally stimulated with added ATP. Fixation withH 2 is consistently lower than with pyruvate. This may, however, reflect a differencein ability of these two electron donors to reactivate an enzyme (as shown for KBH4,

ref. 3).With charcoal treatment it was not possible to decide whether there was a

reaction involving ATP in the pyruvate pathway, although dialysis experimentshave indicated that this must be true3 ,4. If such a requirement existed, it seemedlikely to be met by a much lower concentration of ATP than that required for the H 2

Biochim, Biopiiys. Acta, 99 (Ig65) 4-86-503

ATP AND N2 FIXATION 497

system. The gradual addition of ATP to an extract from which it had been completelyremoved should then restore N 2 fixation with pyruvate as the electron donor beforethat with H 2. Anaerobic passage of an extract through Sephadex G-z5 removed allbut a trace of ATP and Mg2+; when the ATP was replaced in limiting amounts, therestoration curves of Fig. 10 were obtained. It is clear that N2 fixation is restoredwith less ATP in the pyruvate system than in the H 2 system; restoration of acetylphosphate breakdown closely paralleled that of N2 fixation. We therefore suggest thatthere are two distinct sites of ATP action; one between H 2 and ferredoxin, and an­other either between ferredoxin and N2 or between pyruvate and ferredoxin.

In this experiment, the optimum concentration of MgH for the H 2 system

100'pyruvate

1.0

Fig. 10. ATP titration of N. fixation with pyruvate or H. as the electron donor. Assay systems:pyruvate: 150 pmoles sodium pyruvate, 0.05 {lmole CoA and 2/lmoles MgCl•• with other com­ponents as in the standard system. Gas phase: N. or A. H.: standard system minus ATP. with50 pmoles sodium cacodylate and 5 {lmoles MgCl•. Gas phase: 0.5 atm N. + 0.5 atm H., or A.Extract: 4.4 mg protein from an extract passed through Sephadex G-25. 0-0, pyruvate;.-., H •.

(5 mM) was used throughout; for the pyruvate system, which is more sensitive to theMgH/ATP ratio, z mM MgC12 was used to prevent inhibition at the lower ATPconcentration. An essentially similar result was obtained when a ratio of Mg2+ toATP of z .r was used over the same range of ATP concentration, although thepercentage restoration was somewhat lower at the lower ATP concentrations for bothpyruvate and H2•

Removal of ATP from extracts by Sephadex treatment markedly inhibits oreven completely prevents pyruvate metabolism, even in the presence of CoA andMg2+ (Table IV). The system is relatively difficult to study; the optimum Mg2+/ATPratio at saturating ATP concentrations is z :1, but this provides inadequate Mg2+ atlow ATP concentrations. A compromise approach was to determine the effect of ATPaddition with Mg2+/ATP ratios of 2:1 and 5 :1, with the treated extract supplementedwith the same amount of Mg2+ as that added with the lowest concentration of ATP.From the results presented it is clear that the addition of ATP restores the activityfor pyruvate consumption. Since ATP is in some way required for pyruvate metabo­lism, there is considerable doubt whether ATP is required for any reaction subsequent

Bioobim, Biophys. Acta, 99 (1965) 486-503

M. J. DILWORTH ei aI.

TABLE IV

RESTORATION OF N. FIXATION AND PYRUVATE CONSUMPTION BY ATP

Standard assay system, with 0.05 ILmole CoA and 80 f~moles sodium pyruvate. Pyruvate andNH. determined after 30 min. Extract, 2.9 mg protein from an extract passed through SephadexG-25. Gas phase, N. or A. Control activity (I roM ATP, 2 roM MgCl.), 11.4 mumoles N, per mgprotein per min and 238 mftmoles pyruvate per mg protein per min.---------- --- --------_.. -

Addition N.fixed Pyruvate------- [mumoles Img consumedAT? MgCI. proteinjmin] [mumoles /mg(flmoles) protein/min)

0·4 0.0 00.2 0.4- 5. 2 760·5 1.0 9·4- 1561.0 2.0 10.1 195

1.0 0.0 °0.2 La 7·3 1440·5 2·5 9·9 184I.O 5.0 II.O 228

to ferredoxin reduction. Further work on ATP function in pyruvate oxidation is inprogress.

I nhibitor studiesSince it had been proposed- that ATP functioned by activation of a reduced

nitrogenase or that the N 2 molecule was itself activated on the enzyme", evidencewas sought for the occurrence of H 2-dependent acetyl phosphate breakdown wherenitrogenase was either absent or inactive.

TABLE V

EFFECTS OF H., CO AND N.O ON N. FIXATION AND ACETYL PHOSPHATE DEGRADATION IN EXTRACTS

OF C. pasteurianum

Assayed in the standard reaction mixture. Protein added: Set A, 6.9 mg in a final volume of2 ml: Set B, 4.5 mg in a final volume of r ml,

Gas phase N.fi;red Acetyl phosphate used

mumoleslmg % mumolesjmg %protein/min protein/min

-------- ------- -_._-----

Set A10% H. 6% N. 84% A 9.'.2 100 139 10050% H. 6% N. 44% A 5·9 64 185 133

Set B30% H. 30% N 2 40% A 8·5 100 168 100

30% H 2 30% N. 39'% A 1% CO 0·9 10 136 8130% H. 30% N. 38% A 2% CO 0.0 ° 146 87

30% H. 30% N~ 20% A 20% N.o 1.1 13 143 8530% H. 30% N. 40% N.O 0.1 I 136 8T

Biochim, Biopllys. Acta, 99 (1965) 486-503

ATP AND NZ FIXATIO}I 499

Nitrogen-fixing activity is known to be absent from ammonia-grown cells ofC. pasteurianumzo. Extracts of such cells also lack the system for Hz-dependent acetylphosphate decomposition, suggesting that the two functions could be properties ofone enzyme.

A different approach was to use various known inhibitors of Nz fixation to in­activate the nitrogenase. The results of studies with Hz, CO and NzO are presentedin Table V.

Nz fixation was inhibited 36% by increasing the PHzfrom 0.1 to 0.5 atm, whileacetyl phosphate disappearance was increased some 33% (Table V). It appears thatHz was influencing two different systems; one degrading acetyl phosphate (ATP) inwhich Hz acted as a substrate (Fig. 5), and the other that fixing Nz.

CO and NzO were found to inhibit fixation completely at 0.02 and 0-4 atm,respectively; at these pressures there is only a slight inhibition of acetyl phosphatebreakdown (about IS %). It would seem that there must be two distinct systemsinvolved, or that the "nitrogenase" is a multi-functional enzyme of considerablecomplexity.

Function of Mgz+Since Mg2+ is required for the function of acetokinase in this and many other

microorganisms, it seemed likely that the sole function of Mg2+ was the activationof this enzyme. Titration studies with extracts treated with Chelex roo showed nodifferences in the degree of restoration by Mgz+ of acetyl phosphate breakdowncompared to N, fixation. However, this result would be expected if a lack of Mg2+limited ATP generation.

Studies of the effects of different bivalent cations on acetokinase, Nz fixationand acetyl phosphate (ATP) breakdown in extracts treated with Chelex roo gave theresults in Table VI. The situation with Mnz+ was of particular interest; although it wasequivalent to Mg2+ for acetokinase activation, it was only 30% effective for either Nzfixation or ATP breakdown. This observation indicates that Mgz+ plays another role

TABLE VI

EFFECT OF VARIOUS CATIONS ON THE ACTIVITY OF ACETOKINASE, N. FIXATION AND H.-DEPENDENT

ACETYT. PHOSPHATE DECOMPOSITION TN EXTRACTS 01' C. pastel~dam~m

Values relative to MgH = 100. Absolute activities with ~1gH: acetokinase, 3080 mzzmolesacetyl phosphate per mg protein peT min; N. fixation, IT mJ.lmoles N. per mg protein per min;acetyl phosphate decomposition, 175 mzzmoles per mg protein per min. Acetokinase assayed incacodylate buffer (pH 6.7) under Hz; all other assays under 0.5 atm N. + 0.5 atm Hz. FeH

added as H.-saturated solution immediately after addition of extract; a 5-min assay periodwas used for acetokinase because of rapid autoxidation.

Activatingcation

Acetokinase N 2 fixation H2-dependent acetylphosphatedecomposition

None a5 mM Mg2+ 100

5 mM Mn2+ 102

5 mM CaH a5 roM Co2+ 295 mM Fe2+ IS

aroo

31

a4965

aTOO

26a

4368

Bioobim, Biophys. Acta, 99 (1965) 486-503

500 M. J. DILWORTH et al,

besides acetokinase activation, and this is supported by the data with other cations.With the exception of Ca2+, which is inactive for acetokinase, the rate of formationof ATP from acetyl phosphate is in excess of the maximum rate of acetyl phosphatebreakdown observed with Mg2+. As might be expected, any limitation of ATPmetabolism imposed by an ineffective cation is reflected in the N2 fixation observed;the cation therefore can be considered to function in the reaction consuming ATP.It seems probable that the nitrogenase itself does not require Mg2+.

DISCUSSION

The system studied here is qualitatively similar in all respects to that reportedby MORTENSON4 , with clear requirements for ATP, Mg2+, H 2 and ferredoxin. Thequantitative differences are comparatively minor and attributable to differences inorganism and technique. The higher K m for N2 probably was observed in the previousreport because H 2 at a pressure of 0.5 atm competitively inhibited fixation of N2(see Fig. 6). For this reason, the true K m cannot be determined directly, but presuma­bly is approximately 0.037 atm (derived after calculation of K i ) . The apparent K mfor H 2 in N 2 fixation also was lower than that reported by MORTENSON4 (0.10 atmcompared to 0.28 atm).

The decomposition of acetyl phosphate shows exactly the same requirementsas Nz fixation (ATP, Mg2+ , H 2 and ferredoxin) and therefore is assumed to be in thesame electron-transfer chain. This is supported by the strict correlation betweenacetyl phosphate decomposition and N2 fixation; any condition which limits theformer limits N 2 fixation proportionally (Table VI). Alternative acetyl phosphate(ATP)/H2 interactions which have been considered in an attempt to explain acetylphosphate (ATP) decomposition under Hz have been found unsatisfactory. Activationof ATPases or acetyl phosphatase by H 2 appears improbable, as no evidence for suchan activation could be found (Fig. 8). Significant uptake of H 2 is not observed in theabsence of N2, thus eliminating the possibility of pyruvate synthesis (reversal of thephosphoroclastic reaction) for which H 2, acetyl phosphate, Mg2+ and ferredoxin arerequired'<. It seems most probable, therefore, that acetyl phosphate (ATP) is hy­drolyzed in the course of electron transport, and the lack of H 2 uptake suggests H 2

recycling. It is significant that acetyl phosphate ,(ATP) hydrolysis does respond toPHz as if H 2 were a substrate rather than an activator,

The two approaches used to localize the site(s) of ATP action have yieldedessentially the same conclusion. With depletion of ATP by charcoal, it is possible tocompletely inhibit fixation from Hz, while at least 25% of the activity remains withpyruvate as electron donor. As has been discussed earlier, it is likely that what ismeasured as Nz fixation from pyruvate is a mixed contribution from pyruvate itselfand the Hz evolved from it, and that the residual activity after charcoal treatmentmay represent a higher proportion of "true" fixation from pyruvate. An inhibitionof Nz fixation found in reaction mixtures sparged with N 2 could then be explainedby the sweeping out of Hz and prevention of its re-utilization. It is clear that acetylphosphate hydrolysis occurs rapidly when pyruvate is being degraded by extracts,as it has been shown that the acetyl phosphate concentration reaches a steady valuewhile pyruvate is still being metabolized at a constant rate (see Fig. 5, ref. 8).

Biochim. Biopbys. Acta, 99 (1965) 486-503

ATP AND N2 FIXATION 50!

Similarly, when an ATP-free extract is supplied with increasing amounts ofATP, fixation from pyruvate is restored first, suggesting that the concentration ofATP required in the pyruvate system is much lower than in the H 2 system. It alsoplaces the requirement for a high concentration of ATP outside the pathway commonto both systems.

The requirement for ATP in pyruvate metabolism cannot yet be explainedsatisfactorily. It is possibly required for re-combination of a cofactor with an apo­protein, or as an allosteric activator. It seems unlikely that in its absence acetylphosphate accumulates to toxic concentrations, as acetyl phosphate apparently isnot inhibitory at the low concentrations normally detected. However, the very factof ATP requirement at concentrations known to restore N2 fixation (Fig. IO andTable IV) makes it probable that ATP functions in pyruvate-supported N2 fixationby its influence on pyruvate metabolism.

If one site for ATP function is specific for the pathway from H2 to ferredoxin,then evidence is required for enzyme(s) distinct from nitrogenase which utilize ATPonly in the presence of H 2• Ammonia-grown cells, which apparently provide an idealtest system, do not catalyze H 2-dependent ATP consumption, Negative evidenceof this type can be explained readily in terms of coordinate repression of both nitro­genase and the ATP-consuming system, and might be expected if the system for ATPconsumption is specific for the mechanism of N2 fixation.

On the other hand, CO and N20 completely block N2 fixation while ATP is stillbeing rapidly utilized. Furthermore, H 2 itself inhibits N2 fixation but enhances ATPhydrolysis as if it were a substrate in the reaction. These findings indicate that themajor part of the ATP is consumed by an enzyme (system) distinct from thatfixing N2•

Since it is known that neither the reduction of ferredoxin by H 2 and hydro­genase22 nor the ferredoxin-dependent reduction of hydroxylamine's by H 2 is ATP­dependent, the localization of an ATP site between H 2 and ferredoxin is difficult toexplain. Furthermore, the reduction of N2 by NADH is also A'I'Pcdependent", andmust be considered. A scheme which could explain these observations is presentedin Fig. II.

H2

~YdrOgenOsl,; reduced ferredoxin

enzYme~hydrogenase . Mg2. "activated" reduced

H2 • (~l~~erl (" \ ferrjedOXln --N2

/ AlP ADPNADH AlP

pyruvate

Fig. II. Working hypothesis for N, fixation.

In this scheme, H 2 is used to reduce an intermediate carrier (possibly NAD+)with hydrogenase as the catalyst. From the reduced carrier, an activated form ofreduced ferredoxin can be obtained by the action of enzyme "X" and ATP; this formof reduced ferredoxin can be obtained directly from pyruvate. The activated ferre-

Biochim, Biopbys. Acta, 99 (1965) 486-503

502 M. J. DILWORTH et al,

doxin could be degraded either spontaneously or enzymically to the reduced ferre­doxin which is in equilibrium with H 2 through the action of hydrogenase. This schemeis compatible with the direct ATP-independent reduction of hydroxylamine's, andwith the ferredoxin- and ATP-dependent evolution of H 2 from NADH observedby MORTENSON24 • Similarly, it would be compatible with the ATP-dependent evolu­tion of H 2 from dithionite found with the N2-fixing particle from Azotobacter vine­landii25 .

The scheme presented shows no function for ATP at the site of actual N2

reduction, as there is no definitive evidence for such a function. It is possible that the15-20% inhibition of ATP consumption with CO and N20 does represent an ATPinvolvement at this site, but this is highly speculative. It appears that a requirementfor ATP for pyruvate metabolism per se would adequately explain the requirementfor ATP in N2 fixation from pyruvate. Were the ATP required for the same type offerredoxin activation as postulated for the H 2 system, the quantitative requirementsfor ATP in the two systems would be expected to be very similar, but the concentra­tions of ATP required differ substantially.

Therefore, it has been concluded that one site of ATP function is specific to theH2 system for the generation of the form of reduced ferredoxin active in N2 fixation,and that another function lies in the initiation or maintenance of pyruvate metabo­lism. Based on the present evidence, no definite function for ATP can be assigned tothe site of N2 reduction.

ACKNOWLEDGEMENTS

This work was published with the approval of the Director of the WisconsinAgricultural Experiment Station and was supported in part by grant GB-483 fromthe National Science Foundation and by research grant AI 00848 from the Divisionof Research Grants, National Institutes of Health.

REFERENCES

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265·6 D. W. S. WESTLAKE AND P. W. WILSON, Can. J. Microbioi., 5 (1959) 617.7 R. D. DUA AND R. H. BURRIS, Proo. Natl. Acad, Sci. U.S., 50 (1963) 169.8 J. E. CARNAHAN, L. E. MORTENSON, H. F. MOWER AND J. E. CASTLE, Biochim. BioPhys. Acta,

+4 (1960) 520.9 A. W. D. AV1S0N, J. Chem. Soc., (1955) 732.

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Minneapolis, 1964.14 F. LIPMANN AND L. C. TUTTLE, ]. Biol. Chem., 153 (1944) 571.IS M. J. DILWORTH, Biocbim, Biopbys. Acta, 56 (1962) 127.16 I. A. ROSE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. I, Academic

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