Fixation of Atmospheric Nitrogen by Microorganisms By Diethelm Kleiner [*I In Memory of Artturi Ilmari Virtanen

Research on the enzymatic assimilation of molecular nitrogen (Nz fixation) has made decisive advances in recent years. Both the purely scientific aspects of this process and its industrial implications are assuming ever-increasing interest. Some of the results most important from the biochemical point of view have been collected in the present report.

1. Introduction The fixation of atmospheric nitrogen by microorganisms is a process whose importance is exceeded only by photosynthesis. The pioneering contributions to its study are closely connected with the names of D. Burk, P. W Wilson, and A . I . Virtanen, but biochemical elucidation of the enzymatic processes involved took a leap forward only when, in 1960, it became possible to prepare cell-free extracts capable of fming nitrogen"! Atmosphere

Free-living Nz-fixing agents are widely distributed in nature, being found in many soil^[^-*^, in lakes and sea^[^-'^', on Antarctic rocks['41, in hot springs1i51,in the wood of dying Table 1. N,-fixing organisms. a) Free-living 1. Bacteria (e.g. Clostridiuin, Azotohacter) 2. Blue-green algae (e.g. Anahmna. Tolyporhrir) b) Symbionts 1. Blue-green algae in lichens 2. Blue-green algae or bacteria in leaves (leaf symbionts) 3. Rhizobia in leguminous plants 4. Several mainly unknown microorganisms (Actinomycetes?) in the roots of angiosperms

trees['"!and in thegut oftermites~'71,mammals[i81,and man['*l. More important agriculturally and ecologically, however, are the symbionts. particularly the bacteria that occur in nodules on the roots of higher plants.

2 Inertness of the Nz Molecule

Ground water

1*3711

Hydrosphere and lithosphere

Fig. I. Nitrogen cycle in Nature (after [Z]).

Figure 1 shows the nitrogen cycle existing in nature in which part of the inorganic nitrogen present in the soil is washed out into the ground water and a further part is lost to the air by the action of denitrifying bacteria (reduction of nitrate to N 2). These losses are generally balanced by three processes: formation of nitrogen oxides by atmospheric electrical discharges (a few percent), industrial fertilizer production (ca. 30 %), and microbial activity (ca. 70 %)['I. Many of the microorganisms capable of fixing nitrogen remain to be discovered. Those organisms that are known to have this action-whether existing free or in symbiosis-belong exclusively to the domain of the procaryotes (bacteria and blue-green algae) (Table 1). Of the sporadic reports that continually appear about N Z fixation by fungi, not one could be confirmed on reinvestigation by means of the very sensitive acetylene-reduction method (see Section 3.2)[3J. [*] Dr. D. Kleiner Chemisches Laboratorium der Universitat 78 Freiburg, Albertstrasse 21 (Germany)

80

Nitrogen is known to be an unreactive element. Comparison of some of the physical properties and the molecular parameters of the isoelectronic compounds Nz,CO, and C2H2 (Table 2) shows that the main reason for this lack of activity lies in the great increase in bond energy occurring on transition from the double to the triple bond[i91. Table 2. Physical properties of the isoelectronic molecules Nz, CO, and C 2 H 2(after [19]).

Bond length [A] Ionization energy [eV] Dissociation energy [kcal/mol] Bond energy [kcal/mol] of the I st bond 2nd bond 3rd bond

1.098

1.128

1.208 11.4

15.6 225

235

200

38 100

84 170

147

225

235

200

14.0

83

Although the reaction

is exergonic with AGO= - 12 k ~ a l / m o l [ ' ~it~is, immeasurably slow at room temperature even in the presence of industrial iron catalysts (Haber-Bosch process). In industry temperatures ofat least 400°C and pressures of about 200atm are necessary to afford worthwhile conversion[zo! Angew. Chem. internat. Edir. i Vol. 14 ( 1 9 7 5 )

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3. Biochemistry of Microbial N, Fixation

Table 3. Compounds reduced by nitrogenase (after [ 3 9 ] ) n=Number of electrons transferred, V,,I= relative reaction rate.

3.1. Reaction Path

Substrate

In N2-fixing microorganisms the assimilation is catalyzed by the binary enzyme complex nitrogenase. In this process the nitrogen is reduced according to eq. (1) to ammonium ionsL2'], which are probably bound to glutamate with the aid of the

Product

Kd

n

6 2 2 2

N2 NB N2 0 C2H2 HCN

6

1

3

3 3-4 0.6

4

Nz + 8 Ho

+

6 eo T r>

Mg'@

2 NH4@

7-Glutamine (1)

6-15 ATP

C3Hh.

2 He

ADP+ Phosphate

6 6

CHKN CH3NC

'

AT P

enzyme glutamine synthetase'221; ferredoxin is presumably responsible for the electron transport[23-271. As in the HaberBosch process, the reaction can be induced only with a considerable expenditure of energy, which is here made available in the form of ATP[28-321.The number of ATP molecules consumed in conversion of each N2 molecule is not known exactly; in experiments in uitro it changes with the experimental conditions[33-351.

C,Hm

H2

8. 10 12, 14 2

0.2 0.8

4

is the rate-determining step. Kinetic analysis of the mutual influence of the substrates has led to the hypothesis of a flexible active site capable of binding substrates with various structures[411.All the reactions except evolution of hydrogen are inhibited by CO[4'. 421; inhibition of the reaction by the product ADP is of physiological importance[431(see Section 4), but it is not yet clear whether this is due only to product inhibition or also to a regulatory inhibition of the enzyme[39.44* 451. Oxygen denatures the enzyme irreversibly.

3.4. Enzyme Purification

3.2. Determination of the Nitrogenase In the oldest and most direct method the reaction is followed by means of heavy nitrogen. After incubation the tissue or enzyme preparation is subjected to Kjeldahl digestion, the N H 3 is oxidized to N2, and the I5N content of the nitrogen is determined by mass spectrometry. This method is used mainly for the determination of the activity in uiuo. Ammonia can also be measured directly by colorimetry if the enzyme preparations under study cannot induce its further reaction. In such cases the cofactors shown in eq. (1) (Mg2+, ATP, and an electron donor, for which dithi~nite[~'Ihas proved very suitable) must be added to the (usually cell-free) preparations. In the simplest and most sensitive method, which is therefore most frequently used, acetylene is reduced to ethylene, this reduction also being catalyzed by nitrogenase (see Section 3.3). The two compounds can be readily determined by gas chromatography. The method is so sensitive that the fixation activity of no more than two blue-green algae colonies can still be determinedl3*!

3.3. Specificity of Nitrogenase The active site of nitrogenase is not very substrate specific. To date the compounds listed in Table 3 have been reduced in uitro under the influence of the enzyme. If no substrate is added, the enzyme reduces protons and molecular hydrogen is evolved. It is remarkable that the acetylene is reduced only to ethylene[401.In D 2 0 cis-C2H2D2is formed exclu~iveIy[~~]. Except for the complicated reactions of cyanides and isocyanides, the relative reaction rates are in approximately inverse proportion to the number of electrons transferred, and it is therefore assumed that activation of the electrons Angew. Chem. inrernat. Edir. J Vol. 14 ( 1 9 7 5 )

/ No. 2

Denaturation by oxygen provides the chief difficulty in the preparation of pure nitrogenase, so that all the procedures must be conducted in oxygen-free atmosphere (under N2, H2, or argon). discovered that nitrogenase In 1966 E d e n and Le Cornte14h1 is a complex of two easily separable proteins which, in the absence of systematic names, are designated component I (Mo-Fe-protein, molybdoferredoxin, azofermo) and component I1 (Fe-protein, azoferredoxin, azofer). A general purification scheme with the following principal steps has evolved in the course of time: 1) Preparation of a crude extract; 2) Preliminary purification (mainly fractional precipitations); 3 ) Chromatography on diethylaminoethyl cellulose. Elution with solvents of different ionic strengths can lead to separation of the two components in impure form; 4) Further purification of the isolated components (gel filtration, electrophoresis, fractional precipitations, heat denaturation). This separation of the two components and their purification have been described for several microorganisms. However, in each case it has been shown that neither of the two proteins alone is able to catalyze the reduction of N2, even in the presence of all the cofactors. Suggestions that a third protein is necessary for full nitrogenase activity[47.481 could not be confirmed[491.

3.5. Physicochemical Characterization of the Components Table 4 shows the physical and chemical properties of the nitrogenase components of several organisms. In spite of the differing origins of the components, their properties are very similar. The occurrence of molybdenum in component I and the cold lability of component I1 in many organisms are remarkable. In accord with the low isoelectric points the 81

amino-acid analyses carried out so far on I and I1 from all the organisms show a predominance of acidic amino a ~ i d s [ ' ~ 63.641; - ~ ~ * the absence of tryptophan from I1 is remarkable. According to Marchalonis and Weltman[651, the amino-acid composition of components I from C. pasteurianum, K . pneumoniae, A . vinelandii, and Rh. japonicum indicates their evolution from a common precursor; similarly, components I1 from C. pasteurianum, A. vinelandii, and K. pneumoniae may have evolved from a single ancestral protein. Table 4. Properties of the nitrogenase components from microorganisms. Component I1

Component I Molecular weight: 220000

55000 56000

210000 270-300000 216000 218000 200000

-

64000 66800 ~

Subunits: 2x60000 +2x51000 2x60000 ;2x50000

2 x 27500

2x -

4x70000 4 x 56000 2 x 50ooO +2x60000 4 x 50000 2 species

5.5

27500

2 x 33000 2 x 34600 -

4.6 4.7 4

Content of Mo [atoms per molecule]: 1-1.5 1.73 1.8 0 1.54 I .04 0 1.3 1-2 ~

Content of Fe [atoms per molecule] 4 12-18 3 4 22-24 34-38 24 3.5 18 4 29 Content of S Z e [atoms per mo~ecu~e] 4 8-15 3 4 22-24 26-28 ~

20 16.7 26

c . p. c.p. A. v. A. v. K. p. Rh.j

c . p. c . p.

-

Isoelectric point: 5.0 5.2

Ref.

c.p.

~

-

Organism [a]

3 3.82

-

c.p. A. v. A. v. K. p. Rh.j. Chr.

c.p. A. v. K. p.

~521 1541 [551

A. v. A. v. K. p. Rh.j.

Chr.

c . p. c.p. c.p. A. v. A. v. K. p. Rh.j.

A. v. A. v. K. p. Rh.j.

3.6. Combination of the Components from Different Organisms Although the nitrogenase components from different organisms are very similar, they cannot be combined at will to give active complexes. In particular, those that are only remotely related taxonomically show little readiness to cooperate[39,68. 691. For example, neither the combination of I from A. vinelandii (obligately aerobic) with I1 from C. pasteurianum (obligately anaerobic) nor the reciprocal mixture is capable of N, fixatioIP"! Closer relatives give more positive results; for example, all "cross-reactions" of the nitrogenase proteins from B. polymyxa and K. pneumoniae (both facultatively anaerobic) are almost as active as the homologous It is not yet known what causes these differences, because neither the amino-acid sequence nor the tertiary structure of any nitrogenase protein has so far been elucidated.

3.7. Electron Transport and Reaction Mechanism

c.p. c.p.

c.p. c.p. c.p.

while in I1 they are bound in the same way[661.The occurrence of iron and sulfide-sulfur in approximately equal amounts, the characteristic EPR signals (see Section 3.7), the absorption spectra in the visibleand near UV regiond4'], and the evolution of H 2s on acidification classify both nitrogenase components as iron-sulfur proteins[671.Many members of this class, which is widely distributed among the procaryotes, are characterized by very low electron-transfer potentials.

Only very recently have the roles of the two components in the overall process been established, by means of EPR s p e c t r o s ~ o p y ~ ~ ~Fig. - ~ '2~ shows . the EPR spectra of the two components from C . pasteurianum, alone and in admixture, at 13 K, with and without cofactors. It is assumed that the signals at g=4.29, 3.77, and 2.01 belong to half-reduced I, whereas reduced I shows no signal. In contrast, oxidized I1 is assumed not to display any signal but after reduction to show the signals at g=4.3, 2.05, 1.94, and 1.88. Thus, in the presence of dithionite, I exists in the half-reduced form (a) and I1 in the reduced form (b). The signal of the mixture (c) shows weak interactions between the components, but no electron transfer. Addition of MgATP has no effect on Component I

~511 ~521 C591 [531 [541 C55l c561

A

-

+ + +

c.p. A. v. K. p. M. f.

c601 [611 [551 [621

[a] Abbreviations: C. p.-Clostridium pasteurianum: A. v.-Azotobacter uinelandii: K. p.-Klebsiella pneumoniae: Rh.j.-Rhizobium japonicum: Chr.Chromatium spp.; M. f.-Mycobacterium j7auum.

82

377

Jl

r 205.201 194

-9 4.29.

2.01

3.71

-9

It could be shown by Mossbauer spectroscopy that the Fe atoms in I belong to threedistinct groups with differing spectra,

'I

exhaustedg,

43.

R r

f-

MgATP

4

Cold lability: -

1-

+

Component II

Mixture

43

2.05,

I

J9L

I

-9

Fig. 2. EPR spectra of nitrogenase components I and 11 from C. pasteurianum under various conditions. a)-g) see text (after [70]). Angew. Chem. internat. Edit.

1 Vol. 1 4

(1975)

1 No. 2

the spectrum of I (d), but changes the spectrum of I1 (e). Hence it is concluded that MgATP is bound to 11, and this finding has been confirmed by binding 7 7 1 and other The complex of I1 with MgATP is now in a position to reduce I completely with hydrolysis of ATP, whereupon the signal of the latter largely disappears (0. ATP can thus effect electron activation or conformational change of 11, after which electron transfer can take place. When the electron source is exhausted, I1 is oxidized and I is half-reduced, and only the signals of I are observed (g). Accordingly an electron flow as in eq. (2) may be postulated for the enzymatic reduction of N2.

s20,20

-

this signal to the nitrogenase. A M P has no inhibitory activit y14 31.

5. Possible Industrial Applications Implications of the industrial application of Nz assimilation abound in agriculture, environmental protection, and food technology.

5.1. Agriculture and the Environment 5.1.1. Symbionts

I1

ATP

ADP

Although the electron transport in the enzyme complex has been largely elucidated by these investigations, they provide no information about the composition of the active site or the mechanism of the N, reduction. The oldest and most widely accepted proposal[791assumes reduction of the N2 in three steps, by way of diimine and hydrazine to ammonia. However, in spite of intensive search neither of these intermediates could be discovered, and furthermore, neither hydrazine nor diimine can be reduced by nitrogenaselB0,"I. In two other proposed mechanisms the ability of Mo"' and Mo"' to form stable compounds is emphasized[82-19]. Also, in analogy to inorganic N, complexes[82a1.end-on binding of the N2 has been postulated[821. However, none of these theories has yet been supported by experimental evidence"].

4. Regulation of Nitrogenase Activity in Vivo As already mentioned, microbial reduction of N Z is possible only because energy in the form of ATP is consumed for the activation of the unreactive N 2 molecule, so that this assimilation process is very expensive for the organism. A regulatory system has therefore been developed in many N2fixing organisms, in which the nitrogenase synthesis is repressed by NHf[83-901.The extracellular NHf concentration effecting this repression can be very low for many organisms (e.g. ca. 10pmol/l in A. ~inelandii[~']). If there is a n adequate external supply of NHf (and NO?) ions, this crude regulatory mechanism slows down the expensive N, assimilation that is then no longer necessary. Induction of the synthesis by the substrate N2 probably does not occur; indeed, from the standpoint of evolution it would be useless since there is hardly any biotope on the Earth that is free from Nz[891. A second and more sensitive regulatory mechanism, also mentioned above, is based on inhibition of the enzyme by ADP[431. The organism is thus unable to use u p its ATP completely for N2 fixation unless the ADP produced in this reaction is immediately reconverted to ATP. Even though a low level of ATP decreases the rate of the N z fixation, ADP amplifies

['I

Editorial comment: G. N. Sthrauzer will shortly comment upon the most recent views concerning the mechanism of nitrogen reduction in this journal.

Angew. Chem. intemat. Edit. J Vol. 14 ( 1 9 7 5 ) J N o .

2

It has long been known that cultivation of leguminous plants increases the subsequent yield of other crops (green manuring)19z1.This is due to the N 2assimilation by nodular bacteria (rhizobia), which can impart 80-90% of their nitrogen to the host plants[931.To Virtanen in particular belongs the credit for the development of very active strains of rhizobia, which are used for the inoculation of leguminous seeds and which have led to considerably increased harvests. However, it is still unclear what factors enable rhizobia to form a symbiotic association with leguminous plants in particular. Extensive investigations are now in progress[94,9 5 1 on the clarification of this problem, in the hope that it will be possible to induce other plants also to undergo symbiosis with r h i ~ o b i a ' ~the ~ ] ;recent discovery of symbiosis between a non-leguminous plant (Trema aspera) and a bacterium of the genus R h i z ~ b i u r n [is~ encouraging. ~] In addition to leguminous plants there are other N2-assimilating symbioses of ecological importance, e. g. among the lichens and in the roots of some angiosperms and gymnosperms[97*981. In the last case it has not yet been possible to isolate the endophyte (probably an a c t i n ~ m y c e t e [ ~ ~Both ] ) . groups are extremely important for the colonization of washed out, arid soils, as these are poor in readily soluble nitrates and ammonium salts[92.931. 5.1.2 Free-Living Microorganisms

The free-living N2 fixers excrete up to 80 % [ I 31 of their assimilated nitrogen in bound forms, inter aria as NH,I'O0* loll, hydroxylamine derivatives" amino 'O41 Pep tides[1051growth substances[Io6],and fungicides['061. These excretions can in turn be taken up and utilized by higher plants[ 107. 1081 7

In most soils the number of free-living Nz fixers is small in comparison with the number of other soil but it is assumed that, as a result of their wide distribution, the free-living N z f i e r s make a significant, though small, contribution to the fertility of the soil (5-20kg of N per hectare per year, compared with 100-300 from leguminous plant^[^^.^^! This contribution can be increased in two ways : a) by transfer ofthe genetic material for N, fixation ('nif-genes') to soil bacteria that occur frequently; b) by optimization of the living conditions of free-living N 2 fixers. As regards a), it has recently been possible by conjugation to transfer nif-genes from K. pneumoniae to E. coli["O1, which otherwise cannot assimilate atmospheric nitrogen. This success hasgiven rise to great hopes but it has not yet led to large-scale 83

application, and the transfer of nif-genes to human hereditary material. envisaged by some journalists. belongs for the present to the realm of science fiction. As regards b), the number of N2 fixers can be increased by favorable ecological conditions to such an extent that almost the entire nitrogen demand of a biotope can be met by their activity. This is the case if in nitrogen-poor soils the amount of energy required for the assimilation of N 2 is available. This energy is ultimately provided by sunlight and isconserved in the form ofATPeither by photophosphorylation or by photosynthesis (formation of carbohydrates) and oxidative phosphorylation. Bacteria capable of photophosphorylation and blue-green algae find particularly good living conditions in the warm, well-illuminated flooded areas of rice fields" I ] ; their large contribution to the nitrogen balance has been confirmed by measurements of N2 fixation in siru[l12.1 1 3 1,and it has also been found in numerous experiments that the rice yield can be increased by inoculating the fields with these organisms" 4 - ' I 'I.

'

Other favorable biotopes exist in the root zones of some plants if the roots excrete large amounts of organic compounds. (The reduction of 10 mg of N2 requires ca. 1 g of carbohydrate.) The rhizospheres of cane sugar and bahia grass (Paspalum[' II9])have been investigated most widely. These plants excrete so much carbohydrate that some cane sugar fields have been cultivated for more than 100years without nitrogenmanuring, yet without any decrease in yield'' 19]. Attempts have also been made to increase the yields of other cultivated plants (maize, wheat, oats, potatoes, etc.) by inoculating the fields with N 2 fixers, particularly Azotobacter'Io6' 1 2 0 - 1 2 3 ! In most cases the application of these "bacterial manures" has resulted in a small increase in yield, but ironically this is probably ascribable less t o an increase in the N content (because of the energy deficiency) than to the above-mentioned bacterial production of growth substances and fungicides[lo6*"I. Becauses of the effort involved, the use of"bacteria1 manuring" has decreased greatly in recent years[lo6! This technology could, however, attract renewed attention if questions of environmental protection and the dependence of agriculture on industry come to the fore. Only a small part of the nitrogen in artificial manures is utilized by the plants, the remainder being washed out into the ground water[1o9. 24* 2sl. This in turn contributes to nutrient enrichment of the water and its eutrophication. However, nitrogencontaining secretions from microorganisms are released continuously and are probably better retained in the soil. The major problem remains the provision of a cheap source of energy. Jt has already been shown that the nitrogen content of soil can be increased under certain conditions by the addition of straw (cellulose) because of a stimulating interaction between N,-fixing and cellulose-decomposing bacterial126. 1 2 7 1

'

'

It should also be noted that, owing to their high carbohydrate consumption, free-living N2 fixers could be useful in the biotechnological purification of effluents having a high content of organic compounds[I2*.1291. 5.2. Food Technology

Provision of carbohydrates and proteins for the world's population is one of the most urgent problems of our time. Although 84

this is primarily a political problem['301,yet in the long view new methods of providing food must be developed. Here mention will be made only of two unorthodox solutions of the protein problem. In New Guinea there is a race of Papuans whose daily food consists almost exclusively of ca. 2kg of sweet potatoes together with leaves, and in spite of the resulting permanent protein deficiency the district is densely populated and the adults can perform heavy work[' 311. The solution of this riddle may lie in the significant activity of N, fixers recently discovered in the intestinal flora of these people. When investigating an Indian maize mash ("Pozol"), a group of Mexican research workers" 321 found that its nitrogen content increased by 3 0 4 0 " / , during the 5 to lOdays fermentation period customary in that area; they attributed this enrichment to the activity of N2 fixers. This conclusion should be tested by the acetylene-reduction method, but in this connection it is remarkable how widely the fermentation of food is encountered among many non-European peoples['33!

The author is grateful to Pro$ K . Wallenfels for critical perusal of the manuscript and for stimulating discussions. Received: May 27, 1974 [A 37 IE] German version: Angew. Chem. 17.97 (1975) Translated by Express Translation Service, London

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fi

Preparation, Structure, and Reactions of Halomalondialdehydes [ 1

New synthetic

By Christian Reichardt and Klaus Halbritter[*I Dedicated to Professor Karl Dimroth The present progress report provides a survey of the synthesis, structure, and reactions of halomalondialdehydes, including "cyanomalondialdehyde" (diformylacetonitrile),the only pseudohalogen compound so far known in this series. Halomalondialdehydes are reactive compounds, applicable in a variety of ways, and are characterized particularly by their cyclizations with bifunctional compounds. Many of the reactions discussed below can be applied to substituted malondialdehydes other than those specified.

1. Introduction The chemical properties of saturated aliphatic dialdehydes of the general formula OCH--(CH2).--CHO ( n > 1) do not differ fundamentally from those of simple monoaldehydes. for they act as bifunctional monoaldehydes; however, the first two members of this homologous series-glyoxal (n=O) and malondialdehyde (n= 1)-are exceptional, since in the former compound the two carbonyl groups interact conjugatively and in the latter there is a possibility of keto-enol tautomerism. Glyoxal and malondialdehyde can be regarded as the initial members of a homologous series of unsaturated dialdehydes, and it is then useful to distinguish thosc with an even number of carbons from those with an odd number. Unsaturated aliphatic dialdehydes with an even number of carbons and of general formula OCH-(CH=CH),--CHO, being vinylogs of glyoxal, can be assigned to the series of polyene dyes. Unsaturated dialdehydes of the general formula OCH(CH=CH),- l