Plant Physiol. (1984) 75, 304-3100032-0889/84/75/0304/07/$01.00/0

Calorimetry of Nitrogenase-Mediated Reductions in DetachedSoybean Nodules1

Received for publication October 25, 1983 and in revised form January 30, 1984

PETER G. HEYTLER* AND RALPH W. F. HARDYCentral Research and Development Department, Experimental Station, E. I. du Pont de Nemours andCompany, Wilmington, Delaware 19898

ABSTRACF

Heat evolved by isolated soybean (Glyine max cv Clark) nodue wasmeasured to estimate more directy the metabolic cost assocated withthe symbiotic N2 fixation system. A calorimeter constructed bystandard labortory equipment allowed measement on 1 grm of de-tached nodules under a controlled gas stream. Smultaneougas balanceand heat ontput deteations were made.

There was major heat otu by nodules for all of the nitrgeasesubstrates tested (Hf, N2, N20, and C2H2) further establihing the invivo energy cy of biologIl N2 fixatio. Exposure to a shortburst of 100% 02 patially hinctvated nir se to permit c tionsof heat evolved per mole of subsate reduced. The specific rate of heatevolution for H* reductons was 171 ± 6 kilo ries per mole H2 evolvedin an Ar02 atmosphere, that for N2 fixation was 784 ± 26 kiloaloriesper mole H2 evolved and N2 fixed, and that for C2H2 reductio was 250± 12 kialorles/mole C2H. formed. When the appropriae thermody-namic parameters are taken into account for the different subtes andproducts, a AH' of -200 kiocalories per mole 2e7 is shown to beassocated with active transfer ofel by the nhitrogas system.These values lad to a c ed N2 fixation Cost of 9.5 grMS glucoseper gram N2 fixed or 3.8 grams C per gram N2, which is in coseagreement with earlier calculations based on nodular CO2 producti

The biological advantage of symbiotic N2 fixation to a hostplant appears obvious. From the practical standpoint of cropproductivity, however, it has become clear that the advantagesof biological N2 reduction must be weighed against the concom-itant loss of photosynthate needed to support the process.

This carbon cost ofN2 fixation has been estimated by a varietyof in situ plant techniques, giving a rather broad range of valuesfor various legumes and conditions (14, 15, 19). The more recentestimates (7, 10, 21) converge on a range of8 to 12 gcarbohydrate[C(H20)] consumed per g N2 reduced. This approach involvesuncertainties, such as differences between fixing and nonfixingplants in root morphology, nitrate reductase activity, and rootCO2 uptake.

Nitrogen reduction to aqueous ammonia is slightly favorablethermodynamically, but the biological catalyst requires extensiveinput of energy to lower the high energy of activation of thereaction (19). In addition to reductant, the in vitroenzyme systemrequires activation by 4 mol MgATP for each pair of electrons(3, 13). Further, for each N2 reduced, at least one pair of protons

'Contribution No. 3365 from Central Research and DevelopmentDepartment, Experimental Station, E. I. du Pont de Nemours andCompany, Wilmington, DE 19898.

is simultaneously reduced to H2 at similar cost (per pair ofelectrons), presumably as an intrinsic part of the mechanism(13). In the absence of H2 reoxidation, the minimum cost offixation in terms of photosynthate extrapolates to 5 g C(H20)/gN2 fixed. One can validly question, however, whether the ATPrequirement exhibited by the isolated enzyme complex is char-acteristic of the process in vivo.To measure directly the energetics of the nodule, the heat

generated by isolated soybean nodules under various at-mospheres was measured. Heat released was related to nitrogen-ase activity determined by gas analysis. The effective AH of thereduction processes then can be calculated under relatively phys-iological conditions, and realistic fixation and nodule mainte-nance costs estimated directly.

MATERIALS AND METHODSPlant Material. Soybean (Glycine max cv Clark) seeds were

planted in moist vermiculite. Each seed was watered in with 20ml of a Rhizobium japonicum suspension. On emergence, seed-lings were watered lightly once a day with a complete Hoagland-type medium, and 5 to 7 d later transferred to hydroponic vessels,made of 25.4-cm lengths of 15-cm (i.d.) PVC pipe. Each tankheld four plants and was three-fourths filled with a N-free nutri-ent solution and vigorously aerated. About half the plants hadvisible nodules within 5 d; the other halfwere discarded. Nodulesdeveloped to maximum size and activity after 20 to 30 d andwere well adapted to atmospheric levels of 02.The plants were maintained in a growth chamber with a 12-h

photoperiod at 800 ,E m2 s-1 irradiance and day/night tem-peratures of 24°/18C. A hydroponic N-free medium (11) wasfurther modified (Rainbird, personal communication) as follows:2mM KCI, 1.2 mM CaCl2, 0.67 mM Mes (pH 7.2), 0.67 mMMgSO4, 0.5 mm MgCI2, 0.067 mm K2HPO4; trace elements: 80FsM FeEDTA, 3 Mm H3BO3, 1 Mm MnC12, 0.5 ,uM CuSO4, 0.5 MmNa2MOO3, 0.5 Mm ZnS04, and 0.1 M COC12.Rhobial inocum. R. japonicum SR166, a Hup- strain (i.e.

not capable of oxidizing H2) was kindly given us by Prof. R.Maier, Johns Hopkins University. Agar plates of a mannitol-based medium were cross-streaked and incubated 7 d at 28C.The surface ofeach was flushed with 5 ml of20% sucrose, gentlyscraped to give a heavy suspension, then diluted 10-fold withthis solution for use as inoculum on the soybean seeds. Final cellsuspensions were estimated by turbidity to contain I07-I01 cells/ml.Design of Calorimeter. Our requirement was to make meas-

urements on a small number of intact nodules (about 1 g) whichcould be expected to generate at most 0.1 cal/min. The noduleshad to be flushed with a controllable atmosphere.The sample cuvette consists of a cylindrical mini-Dewar (cus-

tom made). Its outer dimensions are 25 mm diameter x 100mm long. The inner chamber is 8 mm in diameter and 60 mm

304

CALORIMETRY OF SOYBEAN NODULES

0.5 mRPOLYETUK

n O.D.

ETHYLENE

NODULES

SILVERED,DEWAR(10ml)

-TWOTHERMOCOUPLES

FIG. 1. Schematic (not to scale) of the calorimetric apparatus.

deep. The closure is a No. 000 1-holed rubber stopper (cut tohalf-length) with thermocouple leads and polyethylene tubingbrought through the center hole and sealed in with epoxy cement.The cuvette is immersed in a controlled temperature bath. To

establish adiabatic conditions sufficiently stringent for the slowrate of heat evolution and to effectively eliminate the heatcapacity ofthe external Dewar wall, we minimized the inside-to-outside thermal gradient by an electronic feedback system (4).By means of a pair of thermocouples and sensitive differential

4

36

3.2

0)

LaJ

_)Da.

2.8

2.4

2

6

1.2

0.8

0.4

06 12 18 24 30 36 42 48 54 60

TIME (min)

FIG. 2. Temperature rise of nodules in the adiabatic system. Tracedemonstrates 02 requirement, partial inhibition by CO, and CN- sensi-tivity of nodule metabolism.

detector and relay, the bath temperature is continually regulatedto track the internal temperature of the cuvette, essentially elim-inating the gradient and hence any significant heat flow from thechamber. The insulation provided by the Dewar constructionresults in a very large thermal time constant which preventspositive feedback instability. By the same principles, one couldmaintain isothermal conditions by electrically cooling the inter-nal chamber. This is expected to have definite advantages.Our apparatus is schematized in Figure 1. The water bath used

2.50

2.25

1-Q

LIJ

CL,

t75

t50

125

0.75

0.50

0.25

6 12 18 24 30 36 42 48 54 60

TIME (min)

FMG. 3. Temperature rise of nodules in the adiabatic system. Traceshows irreversible inhibition by 02 primarily of the CO-insensitive met-abolic component.

305

HEYTLER AND HARDY Plant Physiol. Vol. 75, 1984

6 12 16 24 30 36TIME (min)

| 12X C2H2*4 i

o \

10

9

a

7

6

4

1~! 3

_2

4 I 642 48 54 60 0

FIG. 4. Hydrogen evolution by nodules in absence and presence ofN2. ArO2 = Argon + 20% 02+ 400 ppm CO2.

+12% N20

A

A-

A-' %%

^-s'! X~~~

10 min

TIMEFMG. 5. Effect of alternative nitrogenase substrates on heat evolution

rate of nodules under an AR02 atmosphere. In absence of addedsubstrate, H+ is reduced as confirmed by H2 determinations (not shown).

was a refrigerated Lauda model K-2/R. Fine gauge, plastic-coatedthermocouples (It-18) and the 0.01 differential electronic ther-mometer (T-6) were purchased from Bailey Instruments, and theadjustable electronic relay (Visor II) from Hampshire Controls.The measuring thermocouple was connected to a Kiethley mi-crovoltometer, whose output fed into a Hewlett-Packard re-corder.A low dead-volume gas flow which allowed minimal thermal

losses from well hydrated gasses was required. Low dead-volumespeeded reequilibration when gas composition was changed dur-

ing the run. To minimize thermal losses, relatively low flow rateswere needed; 2 to 4 ml/min was acceptable, yet fairly easy tosample with gas syringes. The gas had to be well hydrated toavoid heat loss by evaporation, thermally equilibrated to thecuvette temperature, and finally water-saturated at this newtemperature.Component gasses were humidified separately with finely dis-

persing water scrubbers which were extensively flushed with thesame gas prior to the run. The incoming gas mix was then passedthrough a 2.5-m coil of 0.16-cm stainless steel tubing immersedin the water bath and then through a final humidifier (a 8-cmtube filled with moist glas beads) before entering the cuvette.Rubber and plastic tubing is permeable to gases, making it

4 8 12 16 20 24 28 32 36 40HYDROGEN FODCD (md/h)

10 I

b9

0

ETH N PR D )

7 ,.J C

4 coo

0.5 1 15 2 2.5 3 3. 4 45 5ENHYDE PRODUCED4nd/h)

(u% RXED)

FIG. 6. Examples of costing runs, relating heat evolution of nodulesto nitrogenase activity. Nitrogenase was selectively inhibited by 02 PUlSeSduring exprments (see text). Each graph shows two separate runs.Measurements with three substates are shown: (a) proton reduction, asH2 evolution under AR-02; (b) C2H2 reduction; (c) N2 reduction, calcu-lated from H2 evolved in air.

306

Ar/02 b

°\++10% air-40 0

\\Io/ _

- o °_0_°~~~~~~~~0---

10

9

a

7

6

E5E4

21-

0

15.0

13.5

12.0

10.5

9.0IN.a,Ic4 7.5 ,

6L0

3.0

15

0.0

3

4.5 ..a Ar/02 citmosphom

CALORIMETRY OF SOYBEAN NODULES 307

impossible to exclude N2 at low flow rates. Hence, the gas lineswere 0.16-cm stainlessGC tubing. Exceptions were heavy walled

13S -a capillary Tygon tubing, used for short flexible couplings, and

0 oL / 2nylon chromatography valves which minimize leakage and deadt2O-InAr-0. volume. Metal-to-glass connections were sealed with epoxy ce-

10S vo ment.W5

/ Sage syringe pumps using glass 50- or 100ml syringes, lubri-cated with a light silicone oil, maintained constant calibrated

0 / flow rates. Peristaltic pumps, because they require Nrpermeable75 -

/ 1 tubing, had to be abandoned.0 o WsJ s - The system was calibrated periodically for zero stability by"O /partly filling the chamber with moistened paper wads and ad-

45 - - justing the zero control of the differential thermometer to mini-30

mize system drift to less than 0.002°C/min.Thermal response of the system was calibrated by means of

s5 - - electrical resistors (of calculated heat capacity) immersed inmeasured volumes of water and connected to a 1.54-v battery.

0 I I I I The effective heat capacity ofthe inner chamber was 0.46 ± 0.014 8 12 1 20 24 28 32 36 40 cal/'C. Standardization of the system was further checked byHYDROGEN PRODUCED Orxh) determining the heat released during acid-base neutralization.

The values obtained were within 3% of the expected AH values.Heat Loss to Gas Flow. Assuming thermal equilibration andso ' ' ' ' full water saturation of the gas stream (ignoring vapor-pressure

ssb / difference between water and dilute buffer) the heat lost to thein Ar-O2-C /gas stream can be treated approximately as added heat capacity.

so / An air flow of 3 ml/min contributes only 0.03 cal/1°C and argon-oxygen mixtures about 0.05 cal/1°C. This was acceptable for ourmeasurements since the heat capacity of the system (sample plus

40 -/ buffer plus chamber) is about 1.75 cal/PIC.

Analytical Methods. Hydrogen determination and N2 moni-3535- °5a - toring were done by GC using a Hewlett-Packard 5710A instru-

0 ment, with a 1.83-m x 0.3-cm molecular sieve column at 70C30 - and a thermal conductivity detector. Ethylene measurements8 25 0° and acetylene monitoring were made on another Hewlett-Pack-

ard 5710A unit with a Poropak N column at 75C and flame20 ionization detector (8). CO2 was measured with a Beckman 864

IR analyzer.Thermal Correction. In the course of these adiabiatic experi-

10..... . . * ments, there was inevitably a temperature rise of at least 2°C and1 2 3 4 5 6 7 8 9 X (rarely) as much as 5C. To coffct for this, a Qlo of 2 wasETYWE PRODJM 1Vmd/h) assumed for all metabolic processes, and data were adjusted to

the initial 23.5°C. This should have minimal effect on slope-dependent values.

I0 ICalculation ofTheoretical Heat of Reaction. The standard heatc (enthalpy) change, AHW, of any net reaction is the algebraic sum

of the heats of formation (AHWf) of all products minus the heatsSO- in aw o_ of formation of all initial reactants, values available from stan-

dard tables (6). These values stricly apply to compounds in their45-s / o specified standard states, and hence only approximate the bio-

logical situation. Fortunately, the AHf values change very little40 - with concentration in the dilute range, and cumulative errors35asL 4from this approximation would be well under 5%.

30 - ~~~~~~~~~~~~RESULT'S82S Nodules were harvested immediately before use and lightly

blotted. Nodules (0.8-1.2 g) were placed into the chamber and20 _ _ moistened with 0.3 ml 0.05 M potassium malate buffered with1S _ 0.1 M Hepes at pH 7.2. The bath and chamber were preequili-

brated to 23.5°C, (approximately the ambient temperature ofthe10 . , . , nodules). After a 1 to 2 min flush of the desired gas mixture, a

0.4 . 1.2 U 2 242.8 &2 &6 4 constant flow (3 ml/min) of gas was n. The system tendedHYOE ,ODJM (md/h) to stabilize within 6 to 10 min after which the gradual rise in

FIG. 7. Examples of costing runs relating respiration of nodules, as temperature was eorded at full scale = 12.0C.CO2 released, to nitrogenase activity in experiments analogous to those A preliminary experiment is shown in Figure 2. There isin Figure 6. Three substrate conditions are shown: (a) proton reduction; measurable heat output by nodules, and temperature increase inin(Figure 6Throdutio substrate conditionsareshown:(a)p nreduction;'air is essentially linear. Anaerobiosis eliminates 90% of the heat(b) CH reduction; (c) N generation while reintroduction of air restores the original rate.

Table I. Summary ofCalorimetric Costing Data

Excess over [CH20J/2e7 [CH20J/2e- TotalObservdTherodynamcs Actvation Stoichiometric Total N Fixation Cost

AH (slope) (,&( ) Cost cost [CH20OJ2eiCH0/N2 C/N

kcal/mol n kcal/2e- g g/greaction

2H (2e) H2 -171 : 6 14 -181 48.4 15.0 64.4 9.0 3.6

CH (2e) C2H -250 + 12 7 -218 57.6 15.0 72.6 10.2 4.1

N2 2NH3 9.3 3.7+ + -784 ± 26 5 -190 50.4 15.0 65.4 9.5 3.82H+ H2

Table II. Supporting Respirometric Data

Observed . EstimatedCO2/mol Equivalent Nitrogenase Cost(slope) ICH2OJ/mol [CH20]/N C/N

g/g

2H+(2e) H2 2.5t±0.3 0.42 10.8 4.3

C2H2 (2e) C2H4 2.9 ± 0.5 0.48 12.3 4.9

N2 (&) NH3 6.1 ±0.1+ + +2. (est. uptake) 1.4 9.0 3.62H+ H2 8.1

CO produced partial inhibition of heat evolution as expected.While CO should largely inhibit respiration of the plant tissue ofthe nodules, it does not inhibit the electron transport system ofbacteroids (1). Cyanide at 3 mm rapidly inhibits almost allmetabolic activity and as expected also the major heat-generatingprocesses.02 at high concentrations is perhaps the most selective inhib-

itor of nitrogenase (2). Figure 3 shows its effect on heat produc-tion of nodules. The transient increase during exposure is ex-pected from similar respiratory behavior (2). Upon return to20% 02, the heat output is dramatically reduced. As will be seen,this is palleled by loss ofnitrogenase activity. The residual heatevolving activity is relatively more sensitive to CO inhibition,suggesting it is in large part host tissue metabolism.

Slopes at various points of the temperature curves were con-verted to cal/h by taking into account the effective heat capacityof the chamber plus contents. In subsequent thermal data, thisheat output rate is directly plotted as the y coordinate. All ratesand activities are based on 1.0 g fresh weight of nodule tissue.

In most experiments, H2 evolution and/or ethylene formedfrom acetylene reduction were simultaneously followed by gasanalysis. Since these rhizobia lack hydrogen uptake activity,evolution of H2 in a Nr-free, Ar/O2 atmosphere represents totalreductive activity (e- flow) of the nitrogenase system. Acetylenecompletely inhibits the evolution. In the presence of saturatinglevels of acetylene, measurement of ethylene produced providesan estimate of total electron flow. Removal of acetylene restoresH2 evolution. Total electron flow remained constant when sub-strates for nitrogenase were changed, although the total electronflow decayed slightly during the time course of an experiment.

When N2 was introduced into an Ar/02 system, H2 evolutionwasdep Figure 4 shows about 70% inhibition, reasonablyconsistent with the observed partitioning of electron flow bypurified nitrogenase which produces one H2 for each N2 reduced(3, 13).

Figure 5 shows the effect of introducing the alternative sub-strates acetylene or nitrous oxide (9, 18) into a nodule systemwhich had been incubating in an atmosphere of20% 02 in argon.

The increasecfheat prodcucti7on with these substrates is consistentwith the thermodynamics of their reduction, as will be discussedlater.Changes from alternating air and Ar/02 were smaller and

often biphasic. This may be due to N assimilation reactionswhich will lag behind and confound the primary reductionprocess, and is being studied.

Metabolic Cost of Nitrogenase Reactions. To separate thenitrogenase-associated heat production from the endogenousmetabolism, successive pulses of 30 s to 3 min of 100% 02 werepresented to the nodules during a calorimetric run, then rapidlyflushed out. After reequilibration, measurements were resumed.This progressively destroyed nitrogenase activity without signifi-cantly altering maintenance metabolism.From a series of such determinations, the slope of heat pro-

duction versus reduction of some nitrogenase substrates wasdetermined. Figure 6, a to c, shows examples of typical deter-minations of this type. CO2 evolution rates and their respectiveslopes were also measured (Fig 7, a-c).These slopes express cal/Mmol of product, from which AH as

kcal/mol is easily obtained. CO2 measurements yield mol C02/mol product directly. Averages ofsuch determinations are shownin Tables I and II. From the intercept value, the maintenancecost ofthe nodules at zero nitrogenase activity can be calculated.This will be discussed.

DISCUSSION

We assume that glucose is the energy source for the nodules'metabolism.We can then write formally balanced equations in which

glucose is used as sole source of reductant for 1 mol of a givensubstrate. Each molecule of glucose reflects 12 electron pairs (ortheir equivalents) transferred in aerobic glucose metabolism. Theintrinsic heat change (AWH) can be calculated strictly from theinput of reactants and output of products, and will be validregardless ofthe complexity ofthe actual pathways involved. Wewill consider several nitrogenase-catalyzed reactions in this con-text.These reactions even in the presence of nitrogenase, do not

occur spontaneously. Their catalysis by nitrogenase requires alarge activation in the form ofATP hydrolysis, apparently neededto effect electron transfer between the component proteins. Theheat released in this activation (which includes the processeswhich formed the needed ATP from glucose as well as the ATP'shydrolysis) will be released as heat. Loosely, activation will showup as gratuitous heat, beyond that thermodynamically accountedfor by the reaction per se.We will treat our measurements of nitrogenase reactions as

representing the sum of a reductive component (minimal bal-anced equation) and an activation component (excess heat ob-served) and attempt to quantitate the relation between them.

HEYTLER AND HARDY 1PI-ant Physiol. Vol. 75, 1984308

CALORIMETRY OF SOYBEAN NODULES

Hydrogen Evolution. In the absence of alternative substrates,nitrogenase catalyzes the transfer of electrons from reductants(ultimately glucose) to protons, with the net effect of hydrogenevolution at metabolite expense. For purposes ofenergy balance,we can write the minimal redox reaction as

1/2 H20 + /12 C6H,206 - /2 CO2 + H2calculated AH = +10.0 kcal/H2

This reaction states that the addition of 10 kcal is required toconvert the substrates to products with 100% energetic efficiency.Our calorimetric measurements (e.g. Fig. 6a) give an average

observed AH of -171 kcal/mol H2 evolved, derived from theslope of heat versus H2 evolution under an argon-oxygen atmos-phere (Table I). Clearly, the overall biological reaction is veryenergy inefficient as shown by the high heat evolution. Despitethe small heat uptake by the reduction process per se, the overallprocess is highly exothermic, liberating a total of 181 kcal/2e-transferred by nitrogenase (H = -181 kcal/2e-). We will regardthis as heat of activation.

Since the AH of combustion of glucose is -673 kcal/mol, thisactivation cost amounts to 181/673 = 0.27 mol glucose/molelectron pairs. The direct reduction cost indicated by the balanceofproducts is VI2 mol glucose/2e-, giving a total cost of0.35 molglucose/mol 2e- transfer (Table I).

Acetylene reduction. Introducing acetylene into an argon-ox-ygen stream increased heat evolution by the nodules (Fig. 5).Calculation shows that acetylene reduction is indeed more ex-othermic then H2 evolution. By analogy to the above discussion

C2H2 + 1/2 H20 + VI2 C6H,206 -*/2 CO2 + C2H4calculated A = -31.6 kcal/mol C2H4

Our observed measurements (e.g. Fig. 6b) yield an average AHof -250 kcal/mol C2H4, hence an activation cost of -218 kcal/2e-. This corresponds to 0.32 mol glucose oxidized for activationand /12 or 0.083 mol for reduction.

Nitrous oxide reduction. As above, a formal reaction can bewritten as

N20+ 1/'2C6H,206-½/2CO2+N2+ ½/2H20calculated AH = -38.9 kcal/mol N20

This is considerably more exothermic than H2 evolution andenergetically similar to acetylene reduction. The heat evolutionchange seen when N20 was introduced into argon-oxygen isconsistent in magnitude with the acetylene response (Fig. 5),suggesting this electron transfer cost is not radically differentfrom the other 2e- processes considered. The reaction productscould not be accurately measured by our methods, so no exactcosting was made.

Reduction of Nitrogen. It has long been known that, when N2is reduced by nitrogenase, concomitant evolution of H2 occurs.This is believed to be intrinsic to the N2 reduction mechanism(3, 13) with a stoichiometry of one H2 evolved per N2 reduced.The minimal overall equation best representing this process

(taking into account heats of hydration and neutalization)N2 + 4H20 + 1/3 C6H1206 2NH4+ + 2HC03- + H2 (g)

calculated AH = -23.8

By using the rate of H2 evolution in air as a measure of N2reduction, exactly analogous costing experiments can, in princi-ple, be run. In practice, the ideal H2 produced N2 fixed ratio of1.0 is only approximated, since a variable fraction of the nitro-genase electron flow tends to go to excess H+ reduction, and thisfraction may change during aging of nodules.Despite these concerns, we did a series of such costing meas-

urements, plotting heat evolution against H2 evolution in air(e.g. Fig. 6c). These gave slopes of -0.784 cal/mol H2 producedand N2 fixed, leading to the calculation:

AH (calculated for reduction) = -23.8 kcal/mol reactionAH measured = -784 kcal/mol reaction

AH for activation = -760 kcal/mol reaction

or -190 kcal/2e- transferred

The reasonable agreement between this activation value andthose for HI and C2H2 reduction (Table I) supports our ther-modynamic treatment.

In the foregoing examples, the metabolic cost of nitrogenasereactions has been separated into the direct reductive componentand an activation component. The carbohydrate required for thereduction is dictated by the stoichiometry of the net reaction.One pair of electrons (or hydrogen atoms) ultimately derivesfrom 1/2 mol water plus 'A2 mol glucose, regardless of actualpathway. This is shown in Table I in terms of grams carbohy-drate, i.e. '1A2 X 180 or 15 g [CH20O/2e-.To this must be added the activation cost, which we empirically

determined, as shown at about -200 kcal/2e-. This is alsoglucose-derived, since carbohydrate is the major storage material(18). In this instance, the total heat of combustion will bereleased, evolving 673 kcal/mol glucose, because no net accu-mulation of intermediates occurs in a cyclic activation process.In terms of glucose required, the expression (AH.K,/-673) aver-aged 0.29 mol glucose/2e-. The glucose costs, as g carbohydrate/2e-, are given in Table I as the activation cost.The sum of these components constitutes the carbohydrate

cost of each 2e- enzymic step (Table I). Extrapolating to the 8e-nitrogen-fixing process gives the values in the last two columns,representing carbohydrate and carbon costs on a basis of gramsnitrogen reduced.

Respirometric Data. During these experiments, repiration wasalso monitored by analyzing the CO2 concentration in the ef-fluent gas as a check on the calorimetric data. These respirometricresults are given in Table II, and agree reasonably well with thecalorimetric data. In the presence ofN2, CO2 liberation was lowerthan expected. We attribute this to carboxylase activity in thenodules, and rates ofCO2 uptake approximtely equivalent to therate ofNH3 formation (S) are assumed. On this basis we correctedthe measured CO2 output under N2-fixing conditions as indicatedin Table II.Maintenance Metabolism. The heat evolved at zero nitrogen-

ase activity can be attributed to the resting or maintenancemetabolism of the nodules. The value for heat evolution at zeronitrogenase was obtained by extrapolating from the data in Figure6. The values from all our H2 evolution and acetylene reductionexperiments average just over 1.9 cal/h .g fresh weight. This canbe converted to about 110 Mmol C02/h-g dry weight, whichagrees reasonably with a recent study of nodules in situ (16).This maintenance level of heat production is approximately 25to 30% of the maximal heat evolution by fresh fixing nodules.The maintenance activity extrapolated from runs made in the

presence ofN2 was consistently higher (2.5 cal/h.g fresh weight).We tentatively attribute this to assimilation-related processes.Since assimilation of exogeneous reduced nitrogen should im-pose similar demands on the plant, this increase need not beregarded as a fixation cost.ATP Cost of Nitrogenase Activation. Our measurements of

heat production associated with activation of the nitrogenasesystem clearly substantiate the high metabolic cost ofdriving thisenzyme. This has been implied by previous estimates of fixationcost (7, 10, 16) and has often been regarded as being far greaterthan theory would predict (3, 12). The present data allow us toquantitate this component more closely. As shown above, the

309

HEYTLER AND HARDY

heat associated with transfer of 1 mol of electron pairs is equiv-alent to the metabolism of 0.29 mol glucose.How much ATP does 0.29 mol glucose provide to the bacte-

roid? In typical cells where glycolytic plus oxidative phosphory-lation should yield 36 ATP/glucose, it would correspond to 10mol ATP, a rather high activation cost. We speculate, however,that in nodules much less ATP than this becomes available tobacteroids. It is known that glycolysis occurs primarily in thecytosol of the plant cells of nodules (17). Although the existenceof ATP-shuttle routes cannot be precluded, it appears plausiblethat bacteroids are dependent upon tricarboxylic acid cycle-linked oxidative phosphorylation as the predominant source ofATP. Further, the P:O ratio in bacteroids (in contrast to free-living rhizobia) appears to be only 2, as the Cyt a-a3 site iseliminated (1). Overall, therefore, as few as 16 mol ATP mightbe available for nitrogenase within the bacteroids for each 1 molglucose consumed by the nodule.The 0.29 mol glucose/mol 2e-, deduced from the heat of

activation, may therefore yield as little 4.6 ATP to the bacteroid.The best enzymological data indicates that electron transferbetween the nitrogenase subunits requires 4 ATP/2e-. Therefore,the costs of fixation currently being measured do not appear tobe overestimates, and the apparent inefficiency of nitrogenase innodules versus in vitro (3) may be related to ATP generation.

Acknowledgments-Frequent dialogue with Dr. R. Rainbird, then Visiting Sci-entist in our laboratory, was most valuable. He also was resonsible for imple-menting our hydroponic culture methods.

Mr. James Keen, long assocated with our N fixation work, contributed experttechnical assistance. We thank Mrs. Beth Haas for help in manuscript prepration.

LITERATURE CITED

1. APPLEBY CA 1969 Electron transport systems of Rhizobium japonicum. I.Haemoprotein P.450, other co-reactive pigments, cytochromes and oxidasesin bacteroids from Nrflxing root nodules. Biochim Biophys Acta 172: 71-87

2. BERGERSEN FJ 1962 The effects of partial pressure of oxygen upon respirationand nitrogen fixation in soybean root nodules. J Gen Microbiol 29: 113-125

3. BURRS RH, DJ ARP, DR BENSON, DW EMERICH, RV HAGEMAN, T LONES,PW LUDDEN, WJ SWEET 1980 The biochemistry of nitrgenase. In WDPStewart, JR Gallon, eds, Nitrogen Fixation. Academic Press, London, pp

56-664. BROWN HD 1969 Biochemical Microcalorimetry. Academic Pre, New York5. CoKmt GT, III, KR SCHUBRT 1981 Carbon dioxide fixation in soybean roots

and nodules. I. Characterization and compon with N2 fixation andcomposition of xylem exudate during early nodule deveopment. PlantPhysiol 67: 691-696

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