Stewart W. Schneller
University of
South Florida Tampa, 33620
II
Nitrogen Fixation An interdisciplinary frontier
For many years chemists considered nitrogen an inert gas and useful as an atmosphere for severely limited chemical transformations. This is not surprising in view of nitrogen's high dissociation energy (225 kcal), high ionization potential (15.58 eV) and the relative inaccessibility of the highest vacant molecular orbitals (two degenerate, antibonding a orbitals a t -7 eV) which render it somewhat intractable to dissociative, oxidative, and reductive (except by highly electropositive metals) processes (1). Consequently, until recently, chemical modifications of molecular nitrogen have been restricted almost exclusivelv to the vigorous Haber process (2). Biological systems, on the other hand, have found nitrogen a molecule amenable to their own life support processes and to their vital function of maintaining a steady-state level of reduced,nitrogen in the biosphere the controlling factor in protein synthesis (5). The process by which various organisms affect this utilization of molecular nitrogen is called nitrogen fixation. The term nitrogen fixation, which is no longer restricted to biological systems, can, in general, be defined as merely the reduction of molecular nitrogen to ammonia, biologically, or ammonia, hydrazine, amines, etc., chemically. The ammonia formed biologically can be returned to the biosphere to be used by other dependent species or retained by the organism in a combined state (e.g., amino acids) for its own growth and possible future ingestion by higher organisms. Biological Nitrogen Fixation
The appearance of nitrogen fixing organisms is believed to have occurred late in evolutionary development since primordial earth is projected to have possessed ammonia as one of the constituents of its atmosphere and, thereby, supplying biological systems with a convenient source of fixed nitrogen (4). Only when the supply of ammonia diminished and sufficient nitrogen appeared (4) did systems evolve which became independent of exogenous ammonia. Although this characteristic does not appear to be specific for one type or class of organisms, systems capable of this phenomenon have been placed in two classifications: asymbiotic or free living microorganisms (e.g., bacteria and blue-green algae) and symbiotic combinations involving, generally, bacteria and the root systems of certain plants (e.g., Rhizohium bacteria and leguminous plants) resulting in the formation of root nodules which serve as centers for associative nitrogen - fixation (5).
Advances in biological nitrogen fixation proceeded very slowly until 1960 when the isolation of cell free extracts from the anaerobic bacteria Clostridium pas786
/
lournol of Chemical Education
teurianum (6) and the aerobic bacteria Azotobacter uinelandii (7), which would affect i n aitro nitrogen fixation, was achieved. Data accrued from studies pertaining to these and other organisms indicates that the controlling factors in nitrogen fixation are a source of reducing power, an electron transfer agent, an energy source, and an enzyme system (nitrogenase or Nzase) which assimilates these activities and provides a center for nitrogen activation. An overall picture of this description is shown in Figure 1.
Figure 1.
Steps believed to b e involved in biologicd nitrogen fixation.
With C . pasteurianum pryuvate oxidation is the apparent reductant while the reducing equivalence for A. ainelandii has remained obscure (5). In the former system a non-heme iron ferrodoxin protein (8) (involving labile sulfide-cysteine-iron interactions (9)) serves as the electron transport agent from pyruvate oxidation to the Nlase center. With A . uinelandii the electron transfer is carried out by a flavodoxin (flavin mononucleotide with no iron-sulfur groups), cytochromes (heme-iron proteins) and an unidentified non-heme iron protein, each acting individually or in concert (5). Though the requirement for ATP in this process is well established, its exact role is still in question (5, 10, 11). Most certainly, ATP hydrolysis provides the energy necessary for the steps (particularly, electron transport) depicted in Figure 1. Unfortunately, i t is difficult to determine the exact energy requirements involved since the individual steps of Figure 1 are not known. Even though NZ 3 HZ 2 NHz is exothermic (values range from A F = -8 kcal (10) to -19 kcal (4)),energy is required (4) for the biological reduction of nitrogen, and more significantly, it is known that N,ase catalyzes ATP ADP Pi (4, 10). Therefore, in order to sufficiently account for the energetic requirements of the overall process, it is reasonable to assume a t least the first step (to the diimide level), and possibly others, are endothermic with subsequent steps exothermic (4). Regrettably, no agreement (4, 10) exists onthe exact stoichiometry of ATP consumed for each mole of nitrogen reduced or electron pair transferred, limiting any conclusions pertaining to the exact energy requirements to speculation. The N,ase enzyme system, which is now available from a variety of organisms (lo), all suggestive of a rela-
+
-
-
+
tively common active site, appears to be an association of between two and four metalloproteins. From the comprehensive data on C. pasteruianum and A . vinela& the principal components are an iron-protein and an iron-molybdenum protein. Their incomplete properties are summarized in Table 1. Although the exact role of each metalloprotein (i.e., whether one activates nitrogen in a metal-nitrogen array and the second delivers the reducing equivalence in terms of electrons or hydrides) is uncertain, omission of one from a suitable in vitro N2-fixingsystem prevents Nz reduction (10). I n addition to reduction of nitrogen Nzme systems promote (1) hydrogen evolution (which occurs in the absence of added substrate or in decreased yield in their presence), (2) hydrogen absorption (possible source of electrons in A . vinelarulii), (3) the exchange of D2 with H D (quoted as evidence for a diimine intermediate (S)), and (4) the formal, stepwise, two-electron reduction of a wide variety of substrates outlined in Tahle 2. With their ability to promote 2-14 electron reductions, it must be concluded that Nzase systems are the most versatile homogeneous reducing catalysts available (10) even upon substrates normally considered toxic to the intact organism. Additionally, each of the substrates reduced (except the proton) possess a triple bond in one of their canonical structures and, with the exception of alkynes, a lone pair of electrons. These observations shed light on the topography and complexing potential of the active site (3). Furthermore, even though no enzymatic intermediates have been detected, formation of cis-1,2-dideuteroethyleneupon N m e reduction of acetylene in DzO is suggestive of side-on alkyne complexation and, possibly, indicative of metal.-N2binding (10). With respect to symbiotic systems little information has been uncovered due to the extreme difficulty in obtaining active cell free extracts. However, a nonheme iron protein and ATP appear to be involved as does a dependence upon oxygen (5). As in the asymbiotic processes, hydrogen evolution also accompanies fixation (5).
well
complexes of uncertain composition Figure 2.
Metals forming stable nitrogen complexes (131.
complexes available from which investigators could draw analogies to rationalize the suspected activation of molecular nitrogen by metals. However, in a synthesis designed to produce [Ru(NHa)a]Clzfrom RuC13 and hydrasine, Allen and Senoff (12) isolated the first metal complex (I) possessing nitrogen as a ligand. IRu(NHdaNn1Cln I
Since that time stable Np complexes have been realized involving low oxidation state transition metals (vide infra) which, interestingly, also form metal carhonyls with carbon monoxide, itself isoelectronic with nitrogen (10,fS) (see Fig. 2). A broad overview of the preparative methods for metal-Nz complexes is given below (IS) with examples in order to illustrate the versatility of Nz as a ligand and the promise of metal-Nz complexes in exploring the N,ase mechanism (particularly, e.g., eqns. (3) and (6)). Reaction of Metol Complexes with o "Mosked" Nz Source: Nitrous Oxide, Azide, etc.
Ir(PPh&COCl
+ RCON1
-
Ir(PPb)n(N$l
(14)
(1)
Nucleophilic Displacement of a Coordinoted Ligond by N z
FeHCl(depe)*
Inorganic Complexes Related to Nrase
chorocterized complexes
+
As many of the early postulates on the function of metals in Nzase were developing there were no metal-N2 Toble 1.
Properties of N m e Protein Froctions (10)
Stobilizofion of Low Volenf Metol Complexes by NZ Following Their Reduction in o Ns Atmosphere
7rm . ~-Protein .. ~ ~ ~ . .
-~
A. vinelandii
C. pasteuriunum
39,000 2-3:2:8 (free CySH) Iron-Molybdenum Protein A. vinelandii C. pasleurianun Molecular Weight 27O,OO&3OO, 000 l6O,WO-ZMI, 000 Mo:Fe:SP-:CySH 2:32:25:37 1-2:15:15:-
Ni(acac)r
Molecular Weight Fe:Sa-:CySH
Toble 2.
Substrates
Substrates Reduced by N20se (10)
Products
Number of electrons involved
NI + A ~ ( ~ - B+ U ) ~ Ni(Nn)H(PEta)l PEt.
(19) (5)
Conversion of o Ligand Possessing the N - N Bonding into Coordinoted N z
+
[Ru(NHt)sNnOI2+
Cra+
-
+
[RU(N&)~N:]~+ Cr'+
(dl) (7)
A noteworthy observation emanating from the N, complex studies and related to the ability of metals to activate Nz was that the coordinated Nz possessed a lower infrared band ( V N , 1900-2200 cm-') than the Raman active free Nz (m, 2330 cm-'). This indicates considerable perturbation of the bond order of NI with resultant electrical asymmetry and suggestive of endVolume 49, Number 12, December 1972
/
787
on bonding of the Nz (1). The infrared stretching frequency of Nz has now become a significant diagnostic tool into the mode of nitrogen bonding to various metals since the position of the band is related to the extent of metal-N2 u-bonding (uide infra) and, possibly, to the degree to whioh the nitrogen molecule is activated toward further reaction (IS). With the infrared data in mind and by analogy to carbonyl complexes, Chatt (1) has put forth a workable description of the type of bonding most likely to be involved in transition metal-Nz complexes (Fig. 3).
Figure 3.
Proposed model for metol.Nn bonding (11.
This description involves an electron donor-electron acceptor synergistic relationship where the highest filled 3gg orbital of the nitrogen, behavingas the donor, forms a o bond with a vacant metal orbital of similar symmetry. Concurrent back donation of metal electrons from filled d orbitals into the antibonding l r , orbitals of nitrogen result in a metal-Nz u bond manifesting itself in a lower bond order for the Nz molecule and ultimately in a lowered stretching frequency for N2 as referred to above. Therefore, from this model alone, it seems reasonable that a transition metal center can be present in Nzase and serve to activate the nitrogen by means of an intermediate resembling a hybrid of structures (11%)and (IIb). + [M N=N o M=N=N] IIa IIb The interaction of the metal d orbitals with nitrogen antibonding orbitals also may account for the reductive capabilities of the Nzase in that they provide a pathway for electron drift onto the nitrogen from the metal which itself may be dependent upon a second metal for its electron source (1). Up to this time, most attempts to exploit the complexed Nz for further reaction generally have resulted in loss of the N2 but inroads into this area are promising and open for further study (IS). From another approach Parshall (lo), employing a benzenediazoninm salt as the activated nitrogen complex and a platinum hydride as the reductive source, has provided support for the two site hypothesis of Nzfixation by the system illustrated below in which the hydride is regenerated in a catalytic fashion.
Non-Enzymatic Nitrogen Fixation
As outlined above transition metals to the right of Group VB are capable of forming stable Nz complexes while transition metals including and to the left of Group VB, and particularly titanium, have shown considerable versatility (2Z) in effecting chemical nitrogen fixation with only the fleeting existance of a metal-N2 complex reported (25). Much of the work in this area, in which low oxidation state metal complexes are generated in the presence of nitrogen, has emanated from the laboratories of van Tamelen (29) and Vol'pin (24). While Vol'pin has realized ammonia from systems employing transition metal halides in the presence of Grignard reagents or alkyl aluminum compounds under high pressures, van Tamelen's approach has produced significant amounts of ammonia utilizing Ti(IV) complexes, sodium naphthalenide and nitrogen a t ambient conditions. Both groups have successfully achieved cyclic processes for preparing ammonia in yields whioh established titanium as a catalytic partner in the overall scheme. An in depth study by van Tamelen of the titanium systems has culminated in the folIowing observations: (1) involvement of Ti(I1) as the active fixing species, (2) utilization of six equivalents of reducing power/Ti(IV) for optimum yields of ammonia, (3) reduction of bound Nz to, possibly, a nitride, and (4) addition of a proton source which liberates ammonia and regenerates the Ti(IV) material. This data is collected in Figure 4 in which 170% ammonia (based on 2Nd Np/
4
-
J./
4~o*kp-
-
Figure 4.
Cyclic pmcesr for Ti(ll1 promoted redudion of
Nz to NHs (27).
NP 2 NHs) was realized. A more practical source of reducing power has been developed by van Tamelen and co-workers (23) employing electrolytic reduction of titanium tetraisopropoxide a3 the Ti(I1) source and yielding in a cyclic, catalytic process 150-300% ammonia (based on N, 2 NH,). A potentially useful extension of the titanium system utilized by Vol'pili and van Tamelen has been the incorporation of the fixed nitrogen into organic derivatives of ammonia as illustrated by eqns. (8-10).
-
+
N,
(CrH&TiClr PhLi 4PhNHs
788 / lournal of Chemical Educdion
pmmura
(M) (8)
+
(CsHa)nTiC12 PhCOCl
+ Mg
N1
PhCN
(72s) (10)
The most important advance in nonenzymatic nitrogen fixation may evolve from the work of Schrauzer, Schlesinger, and Doemeny (38)in which a system of sodium molybdate, 1-thioglycerol, catalytic amounts of hydrated ferrous sulfate, sodium horohydride, and 2000 psi of nitrogen has been found capable of prodncing 3-5 pmole of ammonia. These investigators have postulated that complexation of nitrogen in Nlase occurs at a molybdenum-containing site and is subsequently reduced by the iron protein portion of the enzyme. Newton and co-workers (29), however, have been able to affect the nitrogen to ammonia reduction, albeit to a lesser degree than the Schrauzer system, by an iron pathway void of molybdenum illustrating a need for further study in this area before definite conclusions can he drawn. Conclusion
On the eve of an acute protein deficiency in the world, research directed toward understanding and affecting efficient nitrogen fixation has progressed from three major directions-biological, chemical, and nonenzymatic. Eventually, the findings from each of these will converge on a central point, bringing with them results which will not only explain the elusive process of biological nitrogen fixation but also introduce new means for modifying the N, molecule to meet the nutritional needs of man in areas now incapable of selfsupport without fixed nitrogen. The interdisciplinary approach inherent in this problem has made it one of interesting collaborative endeavor and one which
possesses problems for many areas of science in addition to chemistry and biology. Literature Cited ( 1 ) CXATT. J., P w c . Rov. Soc. B , 172,327 (1969). ( 2 ) LIUM.B., Chcm.Ind. London. 274 (1960). ( 3 ) H m n r , R. W. F., AND BURNS,R. C., Annu. Rsu. Biocham., 37, 331
~----,.
IlORX,
( 4 ) MORTENSON. L. E . in "SUTVBY of Pr~gressin Chemistry" (Edilor: Scow, A. F . ) , Academic Press. Ne-. York, New York, 1968, pp. 131132, Vol. 4. ( 5 ) Musn*r. R., A N D SMITH. D. C., C D O TCham. ~. Ran. 3.429 (1968). ( 6 ) C*RNAHAN, J. E.. MORTENBON, L. E.. MOWER,H. F., AND CASTLE, J. E . , Biochim. Biophys. Aclo, 38, 188 (1960). (7) NICHOLAS, D. J. D.. A N D F I ~ ~ EDR . J., , Nature, 186,735 (1960). ( 8 ) IL*BINowITz,J.C..Aduon. Chem.Ser., 100,322 (1971). (91 Reference (8).p. 333. (10) HARDY, R. W. F., BUBNS,R. C., A N D PABBHALL. G. W.,Aduan. Chom. Say. 100,219 (1971). (11) SOXRAUZER. G . N.. A N D DOEMENI,P. A , , J. A m w . Chem. Soc.. 93,1608 (1971). (12) ALLEN,A. D . , d N D SENOFF. C. V.. C h ~ mCommun.,621 . (1965). (13) ALGEN,A. D.,Ad~c%n. Cbcm.Scr., 100. 79 (1971). P . , * N D KANO,J. W., J . Amm. Chem.Soc., 88,3459 (1966). (14) COLLMAN,J. (15) D I A M A N TA.~ ,A., A N D S m n m w , G. J., Chem. Commun., 469 (1969). (16) H~ssrsorr.D . E , m o T m e ~H. . . J . Amar. Cham. Soc., 89,5706 (1967). (17) BANCROFT. G . M., M A 1 8 , M. J., AND PRATER, B. E., Chem. Commun.,
(20) HIDAI. M . , TOUINABI,K.. UCHIDA,Y., A N D M I ~ N OA,, . Chom. Commun.. 1392 (1969). . N . . A N D T ~ u e sH.. , J . Amer. Chsm. Soc., 91,6874 (1969). (21) A n ~ o nJ. E. E., FECRTER, R. B.. SCRNELLER, 6. W., BOCEE.G.. (22) T A N TAYELZN. GREELEY, R. H.,AND . ~ E R M A R I , B., J . Amer. Chcm. Soc., 9 1 , 1551 (1969). E . E., Adoon. Cham. SIT., 100.95 (1971). (23) TAN TAMEGEN, (24) VOL'PIN,M. E., A N D SHUR.V. B.. Nolure, 209, 1236 (1966) and references oited therein. (25) LATYAEV*. V . N . , VYBAEN~=AY*, L. I.. S ~ RV.,B.,FYODOROY. L. A., m n VOL'PIN,M. E.. J . Organomefal. Chem.. 16,103 (1969). (26) V A N T*M==EN, E . E., m o RDDLER,H . , J . Amer. Chem. Soc., 9 2 , 5253 (1970). (27) TAN TAMELEN. E. E., Aecounls Cham. Rcs., 3,361 (1970). G., AND DOEMEN:,,P. A,. J . Amar. (28) Sc~nnuzen.G . N . , SCHLESINGZR, Chem. Soc., 93,1803 (1971). W . E.. CORBIN.-J.L., SCHNELDEI, P. W., .ANDBVLEN. W. A , . (29) NEWTON, J . Amcr. Cham. Soc..93.268 (1971).
Volume 49, Number 12, December 1972
/
789
