Plant and Soil, Special Volume 1971, 511-524 Ms. SVW-31

T H E C E N T R A L R E A C T I O N S

O F N I T R O G E N F I X A T I O N

by F. J. B E R G E R S E N

Division of Plant Industry, CSIRO, Canberra, A.C.T., Australia

SUMMARY

An outline is given of the general enzymology of the nitrogen-fixing enzyme complex, nitrogenase and thermodynamic considerations of the function of ATP in the reaction are introduced. Some of the implications of these features of the central reactions of nitrogen fixation are discussed in relation to the role of the process in biological productivity.

INTRODUCTION

In this section, in which fundamental processes in nitrogen fixation are being considered, it is my purpose to summarize briefly some properties of the nitrogen-fixing enzyme complex and to examine some aspects of the process which are related to the IBP theme of biological productivity.

There have been a number of recent reviews which give compre- hensive accounts of the biochemistry of nitrogen fixation 6 s 12 13 21 2s 42 46. Since 1960, when consistently active cell-free extracts were first prepared 16, biochemical studies of nitrogen fixation have been concerned with the elucidation of the factors concerned in the reduction of N~ to NH + and the nature of the nitrogenase complex. More recently, attention has been given to other problems such as the relationship between nitrogenases from different organisms 19 a2 and to the control of synthesis of nitrogenase 20 47 4s 50

Biological nitrogen fixation is not an isolated metabolic event. It is integrated with energy-yielding metabolism, electron transport systems and N H + assimilation systems. In all free-living, nitrogen- fixing bacteria the process is growth-linked and is subject to control

512 F . J . BERGERSEN

mechanisms which govern the synthesis of nitrogenase and of other proteins involved in related metabolism. In symbiotic systems, the microbial component may not be actively growing, but the process of nitrogen fixation is dependent upon the provision of snbstrates from the host and is also subject to the action of control mechanisms which may diminish or enhance the nitrogen-fixing activity. All of this needs to be borne in mind while the central reactions are under consideration.

N I T I U ) G E N A S t ~.

E.nz.vmolog), In all nitrogt,n-fixing syst~,ms which ha\'~' been studied, the active

comt)onent has been identified as a complex consisting of two metalloprotcins, neither of which alone has any demonstrable activity. The larger protein, with molecular weights reported between 100,000 and 180,000, contains 1 or 2 atoms of Mo and from 8 to 16 atoms of Ft . Recently this component from .4zotobacter vinelandii has been crystallized and found to have a molecular weight of 270 300,000 with ratios of Mo: Fc: cys tc in - SH: labile S of 1: 20: 20 :15 i t The other protein, with reported molecular weight of 40,000 to 50,000, contains 2 or 3 atoms ~f Fe x3 46 Sulphide. bonds are present and may be inw~lved in the 1)inding of the metals ira both proteins 4o ,51

Nitrogenases in solution are very readily inactivated by ()._,. Both components are affected but the second, smaller protein (the Fe-protein) is the more sensitive. Consequently, careful anaerobic techniques must be used throughout the preparation, purification and assay of nitrogenase components ta

Inactivation of some nitrogenases at temperatures close to 0°C has been reported. D u a and B u r r i s 22 ')a found that nitrogenase from Clostridium pasteurianum was 80% inactivated in 6 h at 0°C. This was later shown to be due to the inactivation of the Fe- protein component 41. However, nitrogenases are more stable at lower temperatures and successful storage of some preparations for periods of weeks or even months has been achieved under liquid nitrogen.

It has been noted that nitrogenases in cell-free preparations from several bacteria have much higher apparent Km (N.)) than whole

THE CENTRAL REACTIONS OF NITROGEN FIXATION 5 1 3

cell systems 9 aT. This may be due to the differential loss of one of the nitrogenase components during preparation of the extracts.

Substrates, inhibitors, and binding sites Nitrogenase requires adenosine 5-triphosphate (ATP) and a low-

potential reductant for activity. These requirements will be discussed in more detail later. When these are supplied, in the absence of substrate, H + ions from the medium are discharged and H2 is evolved. This activity is inhibited by N2 in a competitive fashion a and the N2 is reduced to NH~-. No intermediates are known between N2 and NH4 + nor are analogues of suspected intermediates reduced. However, the exchange between H + in the medium and D2 at low partiai pressures, which is catalyzed by some nitrogenases in the presence of N2, ATP and reductant, may be due to the presence of diimido- and hydrazino-moieties which are bound firmly to the enzyme 3o al a2

A number of compounds which are electronic analogues of N2, inhibit both H2 evolution and N2 fixation. These are all reduced by nitrogenase in the presence of reductant and ATP. Another analogue, carbon monoxide, is not reduced but is a powerful inhibitor of nitrogen fixation, the Kt being as low as 0.2 mm Hg or 1.3 t~M in solution 9. The reducible inhibitors of nitrogen fixation, which may also be regarded as alternative substrates, and the products of their reduction are as follows; minor products are bracketed:

N20 ~ Nz + H20

N~- -+ N2 + NHa

C2H2 - + C2H4

CN- --~ CH4 + NHa + (CH3NH2)

CHaNC -~ CH3NH~ + CH4 + (C2H4 @ C2H6)

There is some uncertainty about the kinetics of the inhibition of nitrogen fixation except in the case of H2 which is unequivocally competitive. Carbon monoxide, for example, has been reported to be both competitive 9 37 and non-competitive la. Some of the un- certainty may be due to the difficulty of interpreting kinetic data for a complex enzyme whose affinity for substrates may be in- fluenced by the relative concentrations of the two components, or

514 F . J . BERGERSEN

alternatively, precise kinetic relationships may hold over limited substrate and inhibitor concentration ranges. A third alternative has been suggested by several workers 13 32 viz that several binding sites are involved. C h a r t et al. 17 have suggested from the study of bi-metallic complexes containing Fe and Mo, that Fe is involved in the binding of N2 by nitrogenase while Mo is involved in reducing the strength of the nitrogen bonds to a point where they are easily reduced. So far, then, it has not been resolved whether the Fe-Mo- protein or the Fe-protein or both are involved in the binding of the substrates.

Practical el/eels o/ lhc ~lalurc o I n.ilro~wnasc

"File involvement of Mo and Fc ill nitrogenasc explains lnany early observations t)f the link l)etween these elements and N2 fixation in symbiotic and free-living systems (e.g. Refs.lOaa). Deficiencies of these elements undoubtedly reduce the accession of N2 into ecosystems and thus provide restrictions upon pro- ductivity. Legumes growing under sulphur-deficient conditions show evidence of a specific effect upon nitrogen fixation 1 and marginal deficiency of sulphur may restrict nitrogenase synthesis because of the relatively high S-content of the proteins.

The sensitivity of nitrogenase to 02 leads to several important practical implications. It is obvious that aerobic systems must posses'; mechanisms for the;'exclusion of O~ from the nitrogen-fixing site. The excessive respiration of nitrogen-fixing Azotobacter cultures is believed to be due to 02 being scavenged from the site by 'combustion' 46 This results in a very wasteful fixation of N2, which becomes more efficient in terms of carbohydrate utilization, when 02 concentration is reduced 46. Additional protection of the nitrogenase of A. vinelandii has recently been described by Oppen- h e l m et al. 43 44. It was found that N2-grown cells contained an extensive internal membrane system to which nitrogenase appeared to be attached. Cell-free preparations which remained bound to fragments of this membrane system were much less susceptible to 02 than were preparations in which the nitrogenase was in solution.

In the symbiotic system of legume nodules, the paradox of the requirement of O2 for ATP generation in the bacteroids and the sensitivity of the nitrogenase to 02 appears to be resolved by an Oz regulation mechanism involving the nodule leghaemoglobin.

THE CENTRAL REACTIONS OF NITROGEN FIXATION 515

This postulated mechanism permits 02 to enter the tissue at a rate sufficient to maintain nitrogen-fixing activity but free O2 concentrations are maintained at very low levels by the respiratory activity of the bacteroids 6

Facultat ive and anaerobic bacteria apparently possess no protective mechanisms and the nitrogen-fixing activity of the cells is rapidly destroyed upon contact with air (e.g. Ref.7).

Conditions of aeration in nature thus clearly exert profound influences upon nitrogen fixation by aerobic and anaerobic free- living bacteria and by symbiotic systems.

The reduction of acetylene to ethylene by nitrogenase has become a widely used assay for nitrogen-fixing activity; its qualitative validity has been well-established (e.g. Refs. 29 a5 86 49). However, quantitative use of this technique presents a number of problems. Most of these are due to the variable relationships found between N2 and C2H2 reduced in living systems. Some of these aspects have been investigated in our laboratory 7. We have concluded that accurate use of the acetylene reduction assay for the measurement of nitrogen-fixing activity depends upon accurate calibration by ni- trogen fixation measurements using carefully matched procedures.

THE ATP R E Q U I R E M E N T OF NITROGENASE ACTIVITY

It is well known from studies with cell-free preparations of several N2-fixing systems, that an adequate supply of ATP is an absolute requirement for the activity of nitrogenase (e.g. Refs. 27 36 as). In the presence of nitrogenase and reductant, ATP is hy- drolyzed to ADP -t- inorganic phosphate (Pd and substrates such as N2, C~H2 or CN- are reduced. If no reducible substrate is present, H + ions are reduced to H2. Several workers have studied the stoichiometry of ATP utilization in nitrogenase mediated reactions. It has been found that the amount of ATP hydrolyzed is pro- portional to the numbers of electrons transferred, irrespective of the products of the reduction. Because of this, it has become customary to express ATP utilization in terms of electron-pairs transferred, since it is convenient to regard nitrogenase-mediated reactions as occurring in two-electron steps, thus -

516 F . J . BERGERSEN

2H + + 2e -+ H2

6H + + N2 @ 6e -+ 2NH3

2H + ÷ C2H,2 + 2e -+ C2H4

M o r t e n s o n 39 and W i n t e r and B u r r i s 58 have ob ta ined values of A T P ut i l izat ion of 4,0 to 4,6 moles ATP per 2~- {i.e. per mole H2 evolved or ½N2 fixed) using a par t ia l ly purified ex t rac t prepared from C, pasteurianum. This is equivalent to 6-6.9 A TP molecules per NH + ion (the form t)rescnt in solution at biological pH). K e l l y 33 c(}ncluded tha t the efficiency of A T P util ization was affected by the ratio (}f the two proteins of nitrogenase.

Thermody~mmic aspects W i l s o n and B u r r i s 6,1 considered t h e rmo d y n a mi c aspects of

N2 f ixat ion by Azotobacter . Their t r ea tmen t was re-examined by B a y l i s s ~, who compared the free energy changes in the following react ions :

aIl{t

glucose (aq) + 60.,. (gas) -- C{):~ (gas) +- 6H~() (liq)

A{;° -- - 6 8 9 k('al

A(; . . . . . 690 kcal (1)

glucose (aq) -~ 4N~ (gas) ~- 6H~O (liq) q- 8H + (aq) --

-- 6C()2 (gas) + 8NH~ (aq)

AG . . . . 160 kcal

AG -- - -112 kcal (2)

In calculat ing AG £or these reactions, certain assumed biological concent ra t ions were used.

Le t us examine the di//erence in energy yield when glucose is oxidized by 02 or N2. Subt rac t ing React ion 1 from React ion 2 we can obta in :

4N2 (gas) + 12H20 (liq) -q- 8H + (aq) =

= 8 N H + (aq) + 60,) z~G = + 5 7 8 kcal (3)

Al though this seems an unl ikely reaction, it can be expressed as two par t ia l react ions:

THE CENTRAL REACTIONS OF N I T R O G E N F I X A T I O N 517

and

4Ne (gas) + t2H2 (gas) + 8H + (aq) = 8NH + (aq)

AG = - -174kca l (Ref. 2) (4)

12H20 (liq) = 12H2 (gas) + 602 (gas) AG (by difference) = + 7 5 2 k c a l (5)

(c] AG ° + 684 kcal; Ref. 2)

Thus, the hypothetical reactions considered by W i l s o n and B u r - r is 54 and B a y l i s s 2 could have predicted that a net energy input of about 72 kcal of metabolic energy mole -1 N H + (from Reaction 3) would be required to drive the N2-reducing reaction and that this would be due to the metabolic provision of reducing power at the level of H2. This possibility was briefly considered b y the former authors 54, and it is in remarkable agreement with the later obser- vations which established that about 6 moles of ATP (equivalent to 60-72 kcal under biological conditions) are hydrolyzed for every mole of N H + produced.

Thermodynamic considerations of intact, living systems are only approximate guides to the energetics of N2 fixation, because of the unknown cellular concentrations of the reactants and prod- ucts. It is more fruitful to consider balances of cell mass for cultures growing with either N2 or N H + as the N-sources. An example of such a s tudy is that of D a e s c h and M o r t e n s o n is with C. pasteurianurn. They concluded that 13 ATP molecules were used for each molecule of N2 fixed (4.3 ATP per 2e or 6.5 ATP per NH +) which is in excellent agreement with the results of cell- free studies with the same organism 39 5a

Thermodynamic considerations may properly be applied to a cell-free system, in which the concentrations of the reactants and products are known. Let us consider a hypothetical experiment, based on data reported by W i n t e r and B u r r i s (Ref. 39, Table 3 and Fig. 5), in which a phosphocreatine-creatine kinase ATP- generating system is used to keep the ATP concentration constant during the reaction. However, instead of using dithionite, whose action as reductant is complex, let us consider a generating system which supplies a constant concentration of reduced ferredoxin, which is the natural reductant for the C. pasteurianum nitrogenase. The conditions, concentrations of reactants and products and the thermodynamic quantities used in the calculations (see Appendix)

518 F.J. BERGERSEN

T A B L E 1

C o n d i t i o n s a n d t h e r m o d y n a m i c q u a n t i t i e s u s e d f o r t h e e v a l u a t i o n of t h e s t o i c h i o m e t r y

of A T P u s e d i n n i t r o g e n f i x a t i o n

T e m p e r a t u r e

p H

Mg2+ A T P * *

C r e a t i n e - P

N ,

F e r r e d o x i n 2-

( C l o s t r i d i u m )

NH,t + t.I~

C r e a t i n e + P~

3 0 ° C (T = 3 0 3 ° K )

6 .0* A G ° ( H +) = 0

10 r a M * *

5 m M A G ° = - - 7 . 0 0 k c a l . m o l e - t ( R e f . a)

25 m M A G ° = - - 12 .80 k c a l , m o l e -1 ( R e f . x4)

1 a t m A G ° = 0

c o n c e n t r a t i o n [ E ° = - - 0 . 3 9 5 V ; n = 2 ( R e f . z6)

h e l d c o n s t a n t

I b y g e n e r a t i n g A G ° = n F A E °

s y s t e m = - - 18.22 k c a l . m o l e - I

1.30 m M a f t e r 30 m i n A G ° -- - - 18.96 k c a l . m o l e - I ( R e f . 1~)

7 .8 tzmulc~ e v o l v e d in 30 m i u in a b s c x m e ~f N2 : L ' q u i v a l e n t to 0 .019

a t m in a v e s s e l of 10 ml a t 30°( ;

A G ° = 0 ( l a t m )

15.5 m M a f t e r 30 r a i n

* T h e p H f o r m o s t e f f i c i e n t A T P u s e 53

** M o s t of t h e A T P will b e M g A T P a n d A G ° is g i v e n f o r t h i s c o m p l e x .

R = 1 .9872 ca l d e g r e e -1 m o l e -x.

F = 2 3 , 0 6 4 ca l g r a m e q u i v a l e n t -x v -1.

A G ° = f r e e e n e r g y of f o r m a t i o n ill t h e s t a n d a r d s t a t e s g i v e n in t h e q u o t e d r e f e r e n c e s .

are given in Table 1. The reaction observed is:

N,, (gas) + 3 ferrcdoxin 2- (aq) + 8H ~ (aq) +

+ 12 creatine-P (aq) = 2NH + (aq) +

+ 3 ferredoxin (aq) + 12 creatine (aq) + 12P, (aq) (6)

The results of W i n t e r and B u r r i s 5a clearly showed tha t the ATP was not used in the reduct ion of N2, since similar amounts were hydrolyzed when N2-fixation was completely inhibited by CO and all of the reducing act ivi ty was expressed as H2 evolution. Reaction 6 should therefore be regarded as the sum of the par t ia l reactions:

6H + (aq) + 3 ferredoxin 2- (aq) + 12 creatine-P (aq) =

= 3H2 (gas) + 3 ferredoxin (aq) +

+ 12 creatine (aq) + 12 P, (aq) (7) and

N2 (gas) + 3H2 (gas) + 2H + (aq) = 2NH + (aq) (8)

In this way, the energy required for N2-fixation can be seen to be a

THE CENTRAL REACTIONS OF NITROGEN FIXATION 5 1 9

consequence of the provision of reducing power in a form suitable for the reduction of N2.

Reaction 7 can also be regarded as the sum of two partial reac- tions :

3 ferredoxin 2- (aq) + 6H + (aq) ~ 3H2 (gas) + 3 ferredoxin (aq) (9)

and 12 creatine-P (aq) = 12 creatine (aq) + 12 P, (aq) (10)

Under the conditions of Table 1, it can be calculated (see Appendix) that z~G(experiment) for Reaction 9 is -- 11.89 kcal and for Reaction 10, -- 180.18 kcal. That is, a total of 192 kcal of energy is expended in generating the H2 (reaction 7) to be used as reductant in Reaction 8.

Evaluation of the ATP generation and hydrolysis under the conditions of the experiment, revealed no free energy discrepancies and therefore the consumption of creatine-P provides a reliable measurement of the energy obtained by reductant-dependent ATP hydrolysis.

Under the conditions of Table 1, AG(exper iment )~ - -22 .14kcal for Reaction 8, if the energy used in providing the H2 is ignored. However, AG(experiment) for H~ production was - -192kca l and therefore the N2 fixation reaction should properly be written:

N2 (gas, 1 atm) + 3H2 (gas, biological, 0.019 atm) +

+ 2 H + ( a q . p H 6 . 0 ) = 2 N H +(aq. 1.3mM) A G = + 1 7 0 k c a l (11)

This expression makes it clear that a net energy input of the order of 85 kcal mole - lNH + is required in this cell-free system and it is in good agreement with the theoretical reactions considered above. Since results obtained with the cell-free system of C. pasteurianum and with chemostat cultures agree in the stoichiometry of ATP use in N,2 fixation is 39 53, it seems reasonable to propose that this expression represents a close approximation to the thermodynamics of the natural reaction of N2 fixation.

I t is interesting to compare this result with the energy consumed in the industrial fixation of N2. Modern large-scale plants, employing natural gas as the source of fuel and H2, have become increasingly economical. M e l d r u m (private communication), has estimated the energy used in such a plant as 11,150 kwh tonne-lNH3. This is

520 F.J. B E R G E R S E N

equivalent to 163 kcal mole-lNH3, or about twice that of the biological system. As in the biological system, most of the energy is used in the production of H2 in a suitable state for the catalytic reduction of N2, which is actually exothermic (c[ Reaction 4).

Practical e]]ects o[ the A TP requirement To some extent, the energy used in obtaining NH + from N2 by

free-living bacteria is not of great importance, since these organisms are found in environments of high C:N ratio and energy supply is not likely to be limiting for fixation. However, symbiotic systems of plants may be at a disadvantage since energy used in N2 fixation would be expected to divert photosynthetic products from being used in growth. Fertilizer manufacturers and salesmen have emphasized this possibility when promoting the sales of their products in several countries. It is worthwhile to consider the magnitude of this effect upon the growth of a legume crop.

Oxygen is required for fixation by legume root nodules (e.g. Ref. 4) and this is believed to be due to the provision of ATP through respiratory pathways in the bacteroids 6. In aerobic bacteria, P/O ratios of between 1 and 2 have been observed 2s but no studies have been made with the non-growing bacteroid form of Rhizobium, nor is it yet clear what is the energy-yielding substrate within the nodule cells 8. However, if we assume a P/O ratio of 2 and glucose as the substrate a4 we can assume that oxidation of glucose will yield 24 ATP mole-I:

glucose + 602 + 24 ADP + 24 P, = 6 CO2 + 6 H20 + 24 ATP

Also, assuming that the ATP requirement for N2-fixation in the nodule system is the same as that for C. pasteurianum (6 ATP/ NH+), the combustion of 1 mole of glucose would yield sufficient energy to allow the production of 4 moles of NH + ; i.e. 180 parts by weight of carbohydrate would be used for the production of 56 parts by weight of N. Expressing this in agricultural terms, in a legume pasture fixing 200 lb N acre -1 annum -I, 640 lb of carbo- hydrate would be used for the provision of the required energy. That is, with a total plant yield of 6-7,000 lb acre -1 annum -I, the carbohydrate consumed in fixing 200 lb of nitrogen could be about 10% of the total yield. Approaching this in another way, if each mole of NH + which is fixed requires about 85 kcal of energy,

THE CENTRAL REACTIONS OF N I T R O G E N F I X A T I O N 521

this is equivalent to 85/690moles of glucose (Reaction 1). Thus, the fixation of 200 lb of N acre -1 requires (200 X 85 × 180)/14 × 690

317 lb carbohydrate. That is, the loss of yield due to the con- sumption of carbohydrate to provide the energy for the fixation of 200 lb of N acre -1 annum -1 could be about 5 per cent of the total yield. This calculation takes no account of the efficiency of coupling of energy-yielding reactions in the nodules and therefore gives a minimum figure.

These calculations have suggested that the energy required for the fixation of N2 by legumes could reduce yield by 5 to 10 per cent. However, it must be remembered that plants assimilating combined N also expend energy in the process and if nitrate forms a sub- stantial proportion of this N, reduction to NH + prior to assimilation into amino acids would be expected to be quite costly in terms of energy because of the diversion of electrons from energy-producing pathways (see Appendix). G i b s o n 24 found no significant differ- ences in relative growth rates when nodulated clover plants assimi- lating N2 were compared with clover plants assimilating N at the same rate from a mixture of NH + and NO{. This showed tha t the N2-fixing plants expended no more energy in obtaining their N than did the plants assimilating combined N. Other effects associa- ted with the production of nodules on legumes may slightly reduce yield, but these are not related to the fixation process.

In aerobic nitrogen-fixing systems, the need to provide both ATP and reductant leads to competition between the terminal respiratory pathways (where ATP is generated) and the electron transport pathways terminating in nitrogenase. This competition is seen, for example, in soybean bacteroids when the respiration rate is lower during active nitrogen fixation than it is after the nitrogenase activity has ceased 6. At low pO2 (atmospheric concentration or lower) root nodule nitrogen fixation is diminished due to limited ATP supply. At high pO2 (0.5 atm or higher for soybean) nodule respiration increases and electrons are diverted from the nitrogenase pathway 4. Aerobic N2-fixing bacteria such as Azotobacter spp. are affected in a similar manner, needing O~ but being inhibited at high concentrations. Thus, as a consequence of the ATP requirement for nitrogen fixation, the degree of aeration of the environment exerts a profound effect upon the rates at which nitrogen is fixed by aerobic systems.

522 F . J . BERGERSEN

ACKNOWLEDGEMENTS

Appreciation is expressed to Professor A. G. O g s t o n and Dr. J. B r o o m h e a d for discussions about thermodynamic aspects of nitrogen fixation and to Dr. F. R. M e l d r u m , I.C.I., A.N.Z., for information about industrial N2 fixation. The provision of a travel grant from the International Biological Programme is also gratefully acknowledged.

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R. Y. S t a n i e r . Vol. I I , pp. 1 58. Academic Press, New York (1961). 26 Hal / , D. O. and E v a n s , M. C., Nature 223, 1342 1348 (1969). 27 H a r d y , R. W. F. and D ' E u s t a c h i o , A. J., Biochem. Biophys. Research Comm.

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THE CENTRAL REACTIONS OF NITROGEN FIXATION 523

33 Ke l ly , M., Biochim. Biophys. Acta 171, 9-22 (1969). 34 K i d b y , D. K. and P a r k e r , C. A., (Personal communication). 35 K o c h , B. and E v a n s , H. J., Plant Physiol. 41, 1748 1750 (1956). 36 K o c h , B. et al., Plant Physiol. 42, 465-458 (1967). 37 L o c k s h i n , A. and B u r r i s , R. H., Biochim. Biophys. Acta 111, 1-10 (1965). 38 M o r t e n s o n , L. E., Proc. Natl. Acad. Sci. U.S. 52, 272-279 (1964). 39 M o r t e n s o n , L. E., Biochim. Biophys. Acta 127, t8-25 (1966). 40 M o r t e t x s o n , L. E. et al., Biochim. Biophys. Acta 141,516-522 (1967). 41 M o u s t a f a , E. and h i o r t e n s o n , L. E., Analyt. Biochem. 24, 226-231 (I968). 42 M u r r a y , R. and S m i t h , D. C., Coord. Chem. Rev. 3, 429--470 (1968). 43 O p p e n h e i m , J. and M a r c u s , L., J. Bacteriol. 101, 286-291 (I970). 44 O p p e n h e i m , J. et al., J. Bacteriol. 101,292-296 (1970). 45 P e n g r a , R. M. and W i l s o n , P. W., Proc. Soc. Exp. Biol. Med. 100, 436-439 (1959). 46 P o s t g a t e , J. R., Nature 226, 25-27 (1970). 47 S o r g e r , G. J., J. Bacteriol. 95, 1721-1726 (1968). 48 S o r g e r , G. J., J. Bacteriol. 98, 56-61 (1969). 49 S t e w a r t , W. D. P. et al., Science 158, 536 (1967). 50 S t r a n d b e r g , G. W. and W i l s o n , P. W., Canad. J. Microbiol. 14, 25-30 (1968). 51 S y r t s o v a , L. A. et al., Mol. Biol. 3, 651-661 (in Russian) (1969). 52 T u r n e r , G. L. and B e r g e r s e n , F. J. , Biochem. J. 115, 529-535 (1969). 53 W i n t e r , H. C. and B u r r i s , R. H., J. Biol. Chem. 243, 940-944 (1968). 54 W i l s o n , P. W. and B u r r i s , R. H., Bacteriol. Rev. 11, 41-73 (1947).

A P P E N D I X

I. Ca lcu la t ion o / t h e t h e r m o d y n a m i c s o / n i t r o g e n / i x a t i o n

REACTION 8.

AGexperlment ~ AG ° + R T l n [N~] . [H2] a . [ H +] 2

A f t e r 30 m i n r e a c t i o n : ( I .3 × 10-3) 2

AGexperiment = - - 37,940 + 1,387 Iog ea l I × (19 × 10-3)3(10-6) 2

= - - 22.14 kca l

REACTION 9.

AG ° + R T l n ( [tt213 i ( a s s u m i n g f e r r e d o x i n AGexpertment \ [H+]6 ] 50% r e d u c e d )

A f t e r 30 ra in r e a c t i o n : (19 × 10-3) 3

AGexperhnent = -- 54,660 + 1,387 log ca l (10-6) 6

- - 11.89 kca l

REACTION 10. ( [c rea t ine]12 . [P,] 12 )

AGexperiment = AG ° + R T l n [ c r e a t i n e - P ] 12

A f t e r 30 r a in r e a c t i o n : (15.5 X 10-2)L°(15.5 × 10-3) 12

AGexperiment = - - 153,600 -[- 1,387 log (9.5 × 10-3) z2

---- - - 180.18 kca l

cal

5 2 4 T H E C E N T R A L REACTIONS OF N I T R O G E N F I X A T I O N

I I . Indus tr ia l n i trogen/ ixat ion

1 11,150 k w h t o n n e -1 = 11,150 × 3 , 6 0 0 × - - - -

4 . 183 1

1 w a t t = 1 j o u l e see -1 - - 4 .183 ca l sec -1

1 × 10 ~ 1 t o n n e - - m o l e s N H a

17

= 8.88 × 104 m o l e s N H a

i.e. N2 ~- N H 3 i n d u s t r i a l l y r e q u i r e s :

11 ,150 × 36 × 102

4 .183 X 5.88 × 104

k c a l

k c a l m o l e 1 N H a = 163 k c a l m o l e - 1 N H a

I I t . Nitrate reduction i~t ptunt,s

NO~" + 10H~ + 8 e - - N H + + 3 ( ) H + 3H~ AG ° = 1 0 5 . 4 k c a l

A s s u m i n g N O r a n d N H + a r e b o t h p r e s e n t in a p t m ) x i m a t e l y m M c o n c e n - t r a t i o n in t h e cel ls a n d a t p H 7.0 a n d 25 ° (298°K) :

AG = - - 105,400 -4- R T l n [ N H + ] [ O H - ] a [H+]a_ ca l FYog~ [H+j~o

- - 105,400 4 1.364 log 10 ~8 ca l

: - - 6 7 . 2 k c a l

l ) i v e r s i o n of 8 e l e c t r o n s p e r m o l e c u l e of NH4+ p r o d u c e d , f r (un r e d u c e d

N A D o r N A D P in t i le t e r m i n a l r e s p i r a t o r y p a t l l w a y of t h e p l a i n s w o u l d be e q u i v a l e n t to 8 - 1 2 m o l e c u l e s of A T P or a b o u t 3 0 - 1 2 0 k c a l p e r m o l e of N H +

u n d e r b i o l o g i c a l c o n d i t i o n s . T h u s , t h e n e t r e s u l t is a c o s t to t h e p l a n t of t h e o r d e r o f 2 0 - 6 0 k c a l p e r m o l e of N H + if t h e e n e r g y o b t a i n e d f r o m t h e r e d u c -

t i o n o f NO~- is c o n s e r v e d a n d c o n s i d e r a b l y m o r e if i t is n o t ,