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Chemical Education Journal (CEJ), Vol. 11, No. 2 /Registration No. 11-7 /Received December 31, 2007
URL = http://chem.sci.utsunomiya-u.ac.jp/cejrnlE.html
Nitrogen Fixation and Dinitrogen Complexes - Revisited
B.H.S. Thimmappa
Department of Chemistry
Manipal Institute of Technology
Manipal University, Manipal, Karnataka, India - 576104
e-mail: bhs.thims manipal.edu
Abstract: The recent advances in nitrogen fixation science and chemical model studies reveal several new phenomena on the
molecular scale. The research study of dinitrogen complexes of transition metals provides an important long-range practical
goal in mimicking biochemical nitrogen fixation processes. In this paper, comparison of natural and synthetic methods of
nitrogen fixation has been presented to have a concise overview. The fundamental aspect of biological nitrogen fixation and
dinitrogen complexes of transition metals with basic background material is presented, using features that enhance the learning
process. This paper emphasizes education-oriented material that is essential for qualitative understanding of the special interest
satellite topic. Development of useful synthetic model systems with significantly more activity for dinitrogen protonation to
produce the product ammonia without wasting precious joules is a key step. Such studies will result in an improved
understanding of nitrogen-fixing processes.An expanding knowledge base in particular areas can provide useful additional
educational information related to a biochemical problem. This entire process of academic study cater to specific interest
groups and help some students to take inspiration from nature in understanding the role of nitrogen fixation with many
attractive features in influencing the nitrogen cycle.
Key Words: Nitrogen Fixation, Dinitrogen Complexes, Nitrogenase Enzyme, Biochemical Conversions, Supportive Education
Introductory Comments
The transformation of atmospheric nitrogen gas to a usable compound such as ammonia, nitrate or nitrogen oxides is called
nitrogen fixation. Nitrogen gas is so unreactive that it is used as a protective inert atmosphere in filament lamps and in the food
industry to prevent oxidation of foodstuffs in cans. Although the earth's atmosphere consists of 78.09 % nitrogen molecules by
volume, most plants cannot assimilate atmospheric nitrogen directly as it occurs in nature (1). The use and intrinsic value of a
good nitrogen fixation education lies in a number of distinctly different fields such as environmental engineering, industrial
processes, agricultural production, nitrogenous fertilizers, bioorganometallic chemistry, genetic engineering, biochemical
technology, nano-biotechnology and educational processes. Nitrogenous compounds released into the environment, their
reactions in the environment are essential and in many cases, have serious implications for the plant and animal life on the
surface of the earth. Nitrogen fixation has been the subject of constant discussion at regular international forums and the 15th
international conference on nitrogen fixation was held at Cape Town in January 2007. Professor G. Ertl was awarded Nobel
Prize in chemistry 2007, for his studies of chemical processes on solid surfaces including nitrogen fixation on metal surfaces
for commercial fertilizer production (2-3). Topics like these with a special focus on relevant information about nitrogen
fixation strengthen students' knowledge base of science by an extra dose of interest and inspiration. The combination of
nitrogen fixation and dinitrogen complexes offers great scope to diversify constant learning experience by imagination and
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association and various interesting aspects could add a different direction for further exploration. This interplay becomes
particularly important in understanding the role of dinitrogen complexes in influencing the risk of environmental effects. Our
goal with this is to develop the overall sense of positive effect both in the regular classroom environment and the popular elearning sector on the net medium that makes more educational sense. There are several valuable internet resources that
provide the related articles with colorful images to browse through and an extensive literature on the educational aspects of
nitrogen fixation that is prevalent in biological systems (4-6).
The prime purpose of this paper is to revisit nitrogen fixation focusing on limited aspects of biological nitrogen fixation and
biomimetic model complexes. It aims to help the reader to learn the basic principles of the process by the explanations or
examples included with facts and figures. Within the unit of study on organometallic cluster chemistry course this special topic
was developed and examined in the regular class with active reception and understanding from the students' community. It can
be used as a valuable auxiliary content in a particular chemical discipline of teaching which provides informative supplement
to students and this can help students to improve their knowledge by extended learning of topical units with straight
connectivity. It may be of practical benefit to young readers who have performed a considerable portion of their background
scientific journey and those in the process of learning in technical educational institutions. The field of study of nitrogen
fixation is obviously a complex and challenging area that can only be surveyed at an introductory-level in a paper of this scope
and for additional details the reader can refer to several excellent primary references and the review articles appeared in
standard international journals (7-15). This introduction to the topic will enable the students to obtain clarification of concepts
related to nitrogen fixation and to explore the details of this fixation process later in their career. It may be useful in intercollegiate environmental awareness program with extended learning value or training in projects to promote organic forming
for sustainable development and growth.
One of the striking changes in the learning trend is the growth of selected section of student learners to be more demanding
consumers of educational activities including an online education in easy-to-access format. In this context, encouraging
exposure to such special topics via network-based education will give them wider vision of the overall process of learning and
open their mind to different possibilities in complete alignment with general learning objectives. The added highlight of this
paper is that the special interest satellite topics continue to cater to specific interest groups of students in a class and motivate
more students towards multidisciplinary and interdisciplinary research approaches. In addition to teacher-directed classroom
learning (in-class learning), self-effort through web-based learning (on-line learning) by students can transform them by
internal awareness process. This is significant when we consider the shift in learning styles from the instructor-based to
information-enabled by web-based instruction. More importantly, with many internet resources and angles, the widely
reviewed topics like nitrogen fixation, fuels the mind of young students to strengthen divergent thinking. While teaching a
course such as bioinorganic/bioorganometallic chemistry, transition metal complex chemistry, metalloenzymes and proteins,
metals in biology and catalysis, small doses of factual knowledge about such relevant learning module facilitate enhanced
learning by students and activate their curiosity and enthusiasm to learn more. This material could form a part of the particular
advanced inorganic chemistry courses such as environmental or atmospheric chemistry to direct the student's interest in the
subject or as an additional activity that can easily fit into regular foundation courses to cement the concepts learnt in the course
and to gain a better understanding of the nitrogen fixation by interaction, information and knowledge sharing process based on
research results.
This paper is organized into fourteen sections; each dealing with a particular compact area in nitrogen fixation. In the
beginning, important aspects including the concerns about imbalance, actual problem of nitrogen fixation, types of nitrogenases
and their characteristic features is discussed to have a coherent narrative through an integrated approach. Synthetic methods,
structural types, bonding aspects, reactivity features in certain representative examples of dinitrogen complexes is presented in
the later sections to provide a glimpse towards the advances that fuel progress. Additional feature is the description of a
chemical extension on nitrogen cycle on the opening pages to assist in extended learning of an important topic of general
interest while the discussions related to heterogeneous catalysis appear at the end of the paper that can have important
implications in the design of better catalysts. An attempt has been made to bridge the gap between the fundamental aspects and
the practical use, essential chemical principles of natural nitrogen fixation and environmentally conscious design of nitrogen
fixation process. A large number of references have been included in the final section to facilitate understanding the subject
matter by further reading.
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Chemical Extension:
Nitrogen Cycle: The cyclic process of circulating nitrogenous compounds between the atmosphere, living organisms and the
soil. This biochemical cycle involves the fixation of gaseous nitrogen into nitrogen containing compounds by bacteria present
in the root nodules of leguminous plants; nitrification of ammonia into nitrite (NO2-) and nitrate (NO3-) salts by nitrifying
bacteria that eventually pass to the plants and animals through the food chain; the conversion of nitrates into gaseous nitrogen
by denitrifying bacteria that enters the atmosphere again completing the cycle (Fig 1).
Fig 1. Simplfied schematic representation of the nitrogen cycle in nature
Concerns about Imbalance
The practical need to improve world food supplies in terms of quality and quantity is one of the powerful motivating factors for
research and development in the various aspects of nitrogen fixation. There is an increasing shortage of natural nitrogen
compounds in the nitrogen cycle that has disturbed the delicate biochemical balance of nitrogen (16). This major concern is
partly due to heavy cultivation of the soil because of increasing world population. The climate change and destruction of
environment due to technical advancement (biomass combustion and fossil fuel combustion) are also contributing factors that
leave a lasting impact. Today, there is a focus on sustainable practices to promote environment friendly culture and even small
steps in the right direction can have big impact on our planet. Large scale destruction of forests that dangerously affect the
ecology is under control with spreading awareness about forest conservation and its importance. The second reason is the loss
of tremendous amount of nitrogen compounds from waste disposal through the sewage drains into the sea that is not easily
available for nitrogen cycle. The downstream movement of waste water to lower locations disturbs the ecology in sensitive
areas affecting the microclimate. The present high level crop productivity is dependent upon the application of large quantities
of nitrogenous fertilizers and the crops produced could have direct macro level implications in improving the nutrition intake of
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local population. However, large scale additional input of chemical fertilizers and pesticides contributes to disruption of soil
chemistry and can cause damage to certain important plant species, thus disturbing the balance of natural nitrogen cycle and
natural ecosystems.
Naturally, population reduction requires long-range planning and comprehensive approach towards education and health care.
Population stabilization and control steps take years for implementation and complex operational parameters are involved in its
focused execution. Another approach is to increase the local adaptation of free-living nitrogen fixing bacteria in soil, a vital bioresource and increased use of specific leguminous plants and other symbiotic associations in agriculture. However, there are
certain constraints in practice due to climate change impacts. The study of the nitrogenase enzyme would define the structurefunction relationship to explain the selective reduction reaction and the possibility of preparing catalytic model to fix nitrogen
on an industrial scale using less expensive reagents. Other significant contribution to fixed nitrogen involves the use of
molecular genetic engineering to certain plants to fix nitrogen for their own requirements as a part of agriculture development
plan. There are a number of known and unknown risks associated with the introduction of genetically modified (GM) crops just
to extend the shelf life of food products, to have protection from insects or to increase resistance to diseases (17-19). The
introduction of transgenic crops for human consumption could contribute to increase the incidence of long-term complications
when compared to outcome of efforts in tune with nature. It is certainly useful to create bacteria ('designer microorganisms')
capable of producing economically important molecules like ammonia and increase the population of such bacteria obtained by
genetic modification method. Biotechnology can be used to facilitate the production of useful protein to protect substrates from
environmental effects/to deliver reactants to the inside of enzyme for actual conversion and subsequent release of the end
product. Also, transfer of human genes to food animals or animal genes into food plants raises ethical concerns and transgenic
foods, though generally regarded as safe, the most reliable analysis due to lack of conclusive toxicity evidence is a serious
matter. The transfer of nitrogen fixing (nif) genes from the relevant bacteria into suitable plants can be of great help to
naturally nitrogen-deficient soils. Alternately, the oxidation of dinitrogen with atmospheric oxygen using homogenous or
heterogeneous catalysts could contribute to the development though environmental stresses pose problems.
A combination of all these possible approaches can improve the health of the peoples worldwide while protecting the
environment and restore original balance in the nitrogen cycle. The efficient use of integrated nitrogen management practices
can further help to optimize nitrogen fixation and a means of ensuring proper food supply to accommodate population growth
(15). The negative consequences of human-induced climate change that raises vital questions about sustainable development
are arrested to some extent by serious forestation efforts all over the world with the resultant rise in the number of trees and
their biodiversity. The recent popularization of organic forming practices has increased soil fertility to a large extent. The
public sector units (PSUs), world conservation union (WCU), non governmental organizations (NGOs), environmental support
groups (ESGs), public-private consortium (PPC), centre for global development (CGD) and self-help groups (SHGs) in many
countries have identified thrust areas and have come up with detailed action plan for the future in the implementation of the
green project in protecting the vanishing rainforests and creating new forest ecosystems. The emphasis on environmental
science education and awareness among people on nitrogen fixation and the green initiatives in industrial progress in the recent
past reflect the growing concern about long-term consequences. The changes in policies and execution methodologies of
governance including integrated projects through public private partnership (PPP), creative use of biomass and water resources,
specially designed seed conservation programs, soil and waste conservation for sustainable forming practices, mega projects to
boost agricultural production worldwide with proper monitoring mechanism highlight the real deep concern about imbalance.
An intense public awareness building initiatives and training programs for younger generation about various aspects of
nitrogen fixation are the need of the hour as a part of the clean development mechanism (CDM). The Nobel peace prize 2007 is
awarded to intergovernmental panel on climate change (IPCC) for extensive research work to combat climate change and to
translate the results of rigorous scientific analysis of climate data into policy matters with its strong technological and
economic focus. The knowledge value of research culture as reflected in research seminars and sessions also partially explains
the increasing interest among teachers for an intense effort to bring such subjects in lectures at the various programs including
online educational programs (OEPs).
Preliminary Considerations
Nitrogen fixation is accomplished either by natural processes that include biological, incidental, photochemical processes or by
industrial processes that predominantly include Haber, arc and cyanamide processes (Scheme 1). The biological process
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contributes the principal component of the overall annual fixation (60%) and involves chemical activation method by using
biological catalysts. The biological fixation is performed only by selected microorganisms that are either free-living (e.g.
Azotobacter, C. Pasteurianum) or by certain associative microorganisms (e.g. Rhizobium bacteria) in contact with plant roots
like cloves, soybeans and blue-green algae. The other two nonbiological processes of N2 oxidation by nature (i.e. lightning and
UV/cosmic radiation) can make a significant impact on agricultural production in certain local areas by providing free fixed
nitrogen to the farmer. Among the industrial processes, the Haber process is the most important one with world production
capacity of ammonia 1.25x108 tons per year. Despite several technological developments in the process, it requires high
temperatures and pressures. The other two oxidative fixation processes are not commercially viable today because of large
energy requirements and as they involve physical activation method by using electric discharge or the use of high temperatures.
The N2 oxidation by an electric discharge process could be used locally where cheap electricity is available and small-scale
production would be sufficient to meet the requirements. The reaction of atmospheric nitrogen and oxygen during the
combustion process (e.g. internal combustion engines [ICE] and thermal power plants [TPP]) generates gaseous nitrogen
oxides, NOX. Today, the nitrous oxide formed by microbial action and as a byproduct of combustion is considered one of the
greenhouse gases causing environmental concern. The synthesis, characterization and application of transition metal complexes
that are capable of dinitrogen reduction in protic media on a laboratory scale also contribute very small quantities of fixed form
of nitrogen at the moment compared to the global input of fixed nitrogen into the environment.
Dinitrogen Fixation
Natural Processes
1. Biological Nitrogen Fixation (BNF)
Direct assimilation of atmospheric
nitrogen by some micro-organisms
R.T. atm.
N2 +
+ 8e2 NH3 + H2
N2ase
2. Incidental Electrical Fixation
Formation of oxides of nitrogen from the
action of lightning in the upper
atmosphere by electrical discharges
8H+
lightning
N2 + O2
2 NO
2NO + O2
2NO2
3. Photochemical Nitrogen Fixation
Oxidation of nitrogen in the air brought
about by UV light or sometimes cosmic
radiation at high altitudes
hν
N2 + 3H2O
2NH3 + 3/2O2
Artificial Processes
Industrial Nitrogen Fixation (INF)
Commercial production of ammonia by
Haber-Bosch process
450° C, 250 atm
2 NH3
N 2 + 3 H2
Fe/K2O/Al2O3
Birkeland-Eyde Process
Dinitrogen oxidation to generate nitric
oxide by electric-arc process
3000° C
N2 + O2
2 NO
Electric discharge
Frank-Caro Cyanamide Process
Calcium carbide reacts with nitrogen to
give calcium cyanamide
CaC2 + N2
1100° C
CaNCN + C
Scheme 1. The chart on the role of the nitrogen fixation by natural and artificial processes
Problem of Dinitrogen Fixation
The reduction of the very inert dinitrogen involves acceptance of six electrons to be converted to ammonia, a multielectron
process. There are certain difficulties with this reduction process to occur under mild conditions. First is the high strength of N
N triple bond (bond dissociation energy 942 KJmol-1). The exceptionally low reactivity of dinitrogen is reflected in its high
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ionization energy (1,402.3 KJmol-1) and low electron affinity (-7 KJmol-1). The ground state electronic configuration of
dinitrogen is (1σg)2(2σ*u)2(1πu)4(3σg)2. There is a large energy difference (2209.5 KJmol-1) between the highest occupied
bonding molecular orbital (HOMO-3σg) and the lowest unoccupied molecular orbital (LUMO-2πg). This makes the dinitrogen
resistant to electron transfer into its vacant π* acceptor molecular orbital and hence the need for greater activation. The
disability of dinitrogen to behave as a π* - acceptor is due to the lack of bond polarity and because of its higher ionization
potential it is a poor σ-donor as well. Further contributing factor leading to low reactivity is the unusually high strength of the
first of the three bonds (418 KJmol-1), which is about half the triple bond dissociation enthalpy. The key step in dinitrogen
fixation is breaking the first nitrogen-nitrogen bond that requires large activation energy.
The simplest mechanism for dinitrogen reduction to ammonia would require three-two electron transfer and protonation steps
to give first diazene (HN=NH), then hydrazine (H2N-NH2) and finally ammonia (2NH3). The energetics of reduction process
involving intermediate stages of diazene and hydrazine indicate that the formation of these two compounds is
thermodynamically unfavorable although the overall reduction of N2 to NH3 is thermodynamically favorable (free energy = -50
KJmol-1). Thus, we have to overcome the strongly endothermic addition of the first two electrons by using very strong
reducing agents. These are the prime reasons why dinitrogen activation is a longstanding challenge to those involved in
research and development in this field. Further, the complexity in complexation and reduction sequence of natural enzyme
systems has been a problem in the design and development of an effective artificial metalloenzyme. There are several cubanetype Fe4S4 clusters that are considered as structural models, but most of these clusters lack in their dinitrogen binding capacity
and subsequent reactivity features to define them as functional models (20-21). The biological nitrogen fixation (BNF) occurs
at soil temperature and atmospheric pressure whereas the industrial nitrogen fixation (INF) requires high-temperature and highpressure to do it. The recent developments in N2 activation by transition metals indicate the possibility of synthetic alternative
to in vivo nitrogen fixation and the prospects of finding an efficient catalyst to replace the Haber process in industry.
The Three dinitrogenases
The dinitrogenases (N2ase) are a class of metalloenzymes with an active center that perform the eight-electron reduction
reaction of equation (1) under aqueous environment.
N2 + 16MgATP +
8e-
+
8H+
N2ase
2NH3 + 16MgADP + 16Pi + H2 (1)
(Pi = inorganic phosphate). The enzyme catalyzes the reduction of dinitrogen to ammonia at moderate temperatures (~300 K)
and at pH values around neutral (pH ~ 7). The sketch summarizing the interrelated processes involving dinitrogen reduction
enzyme is depicted in Scheme 2. There are three types of N2ases distinguished by the metals that they contain [Mo-Fe]-, [VFe] - and [Fe]-nitrogenases (22). Both the Mo & V -N2ases, are expressed by some organisms and the alternative enzymes act
as a "backup system" in case of low [Mo] in the environment. All three N2 ases are extremely sensitive to irreversible
inactivation by dioxygen, thus requiring anaerobic environment for their catalytic activity. The second requirement is a
substantial energy input in the form of magnesium adenosine triphosphate (MgATP) and the third, a readily available source of
low potential (< - 400 mV) organic reducing agents such as naturally occurring ferredoxins and flavodoxins. These key factors
influence the extent of nitrogen fixation by nitrogenase enzyme system. In the conversion of N2 to NH3, 16 molecules of ATP
are transformed to ADP (adenosine 5/-diphosphate), making the enzymatic fixation an energy consuming process (∆Go = -30.5
KJmol-1 for ATP hydrolysis) . The action of N2ase always produces dihydrogen (H2) with N2 reduction, although the
elementary mechanism of its formation is not adequately established. The electron transfer process takes place from ferredoxin
to the Fe-protein, then to MoFe-protein and finally to the substrate as shown in the Scheme 2 and as the substrate is reduced,
Mg-ATP that binds and dissociates to the Fe-protein is hydrolyzed to Mg-ADP. Further, the ammonia produced in the fixation
process should be assimilated efficiently into amino acids and nucleic acids.
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Scheme 2. A schematic diagram of overall electron transport sequence of nitrogenase reaction in nature
Apart from the physiological substrate, dinitrogen, a variety of other small substrates including those containing double or
triple bonds can be reduced by the enzyme; [azide ion (N3-), nitrous oxide (N2O), acetylene (C2H2), cyanide (HCN), protons (H
+), cyclopropene (C3H4) and isocyanides (CH3NC)]. The [Mo-Fe]-N2ase reduces acetylene (C2H2) to ethylene (C2H4) whereas
the [V-Fe]- and [Fe]-N2ases can reduce acetylene up to ethane (C2H6) and this reflects some differences in binding and
reduction for the various substrates. The reduction of acetylene to ethane [Acetylene Reduction Assay (ARA)] is often used as
a test for the presence of the [V-Fe]-nitrogenase (23-28). Thus the various N2ases can also be distinguished by their product
specificities. Moreover, [Mo]-nitrogenase is less effective in N2 reduction at lower temperatures than [V]-nitrogenase. The
ultra-high resolution X-ray structure of the MoFe protein of N2ase indicates the possible presence of nitrogen atom in the
MoFecofactor (29).
The reaction (1) is catalyzed by molybdenum nitrogenase, [MoFe]-nitrogenase. The active N2ase enzymatic complex consists
of two metalloprotein components both of which are required for its activity: Component I: the Fe protein (dinitrogenase
reductase) has a molecular weight of ca. 60,000 and contain a single 4Fe:4S cluster center and probably has an electron
transfer function to the larger protein. Component II: the MoFe protein (dinitrogenase) has a molecular weight of ca. 220,000
and contains approximately 2 Mo atoms, 30 Fe atoms and 30 acid-labile sulfides (S2- units). The MoFe protein contains two
distinct types of clusters: the FeMo cofactor, FeMoco [1 Mo, 7 Fe and 6 S2- units] which is believed to be the metal centre at
which dinitrogen is activated and transformed and the P-clusters that are two 4Fe-4S clusters bridged by cysteine thiolate
ligands, which probably act as electron reservoirs (8-15). Further, two molecules of MgATP undergo hydrolysis in each cycle
and an electron transfer from Fe protein to the MoFe protein takes place in this key biological process, much like one-way
traffic. The reduction probably occurs one electron at a time and the pathway of electron transfer within the MoFe protein is
from the P-clusters to FeMoco. The essential steps in BNF include the following; (i) binding of N2 by the enzyme to induce
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chemical reactivity (ii) electronic activation of the dinitrogen ligand (iii) protonation of coordinated N2 to NH3 (iv) liberation of
reduction product NH3 and (iv) regeneration of the active nitrogenase enzyme complex.
Subsequent biochemical studies on purification and isolation of N2ase from certain type of nitrogen-fixing organisms revealed
the absence of molybdenum and the presence of vanadium nitrogenase, [VFe]-N2ase (23-28). A few years later yet another
type of N2ase which lacks both Mo or V and containing iron was purified from some nitrogen-fixing organisms {iron
nitrogenase, [Fe]-N2ase} (29-32). The smaller component in conventional molybdenum nitrogenase is encoded by nifH gene,
that in alternative vanadium nitrogenase by vnfH gene and the corresponding gene in iron nitrogenase anfH. The larger
component in Mo-N2ase is encoded by nifD and nifK, while that component in alternative enzymes is encoded by vnfD/anfD
and vnfK/anfK. The other two alternate vanadium and iron nitrogenases show the same general structural characteristics, but
subtle differences do exist. For example, the structure of the VFe proteins and FeFe proteins show an additional small subunit
essential for activity. The hybrid protein formed from reconstitution of FeVco and cofactor-free MoFe protein continues to
catalyze the reduction of acetylene and protons, but incapable of dinitrogen reduction (21-25). The reason why specific set of
metals is used by the different N2ases has to be established by physiological studies. The VFe and FeFe active sites are less
active catalytically than the MoFe center. These recent findings raise questions about the possible unique role of only Mo in
BNF. The VFe protein also contains two components: the FeV cofactor, FeVco and the P-cluster, but the identification of
components of the FeFe protein remains unclear. The subtle differences in the type of interaction between N2 and Fe or V in
different nitrogenase systems have to be established by experimental observations and the reduction mechanism may be
different in these three types of N2ases. The action of [Mo]-nitrogenase does not release free hydrazine whereas a small
quantity of it is formed as a product during dinitrogen reduction by [V]-nitrogenase. The interconversion and inversion in
metalloproteins may play a role in N2 transformation so does indirect secondary bonding interactions like hydrogen bonding
through amino acids. The discovery of other characteristics of alternate N2ases to understand nitrogen fixation provides
prospects for their future exploitation. In these different two component systems the mechanisms of substrate binding,
activation and reduction are not yet fully understood.
Dinitrogen Complexes
Modeling the nitrogen fixation will provide insights into various structures, bonding modes which in turn will help to find the
solution and to develop application specific functional models such as polymerization and isomerization reactions. The
discovery of the first dinitrogen complex, [Ru(NH3)5N2]2+ and subsequent synthesis of the first dinitrogen complexes
containing Mo, Fe, V and the first protonation of coordinated dinitrogen are the major milestones in the chemistry of nitrogen
fixation (38-41). The dinitrogen complexes act as a bridge between the BNF and the INF. The study of N2 complexation by
transition metals as synthetic analogues of enzymatic process has proceeded in two fronts. One area is structural models and
the other area functional models. The structural models of the enzyme attempt to mimic electronic features, oxidation states,
internuclear distances, bond angles, dinitrogen binding modes, "lock and key" binding and overall stereochemical
characteristics whereas the functional models of N2ase represent catalytic reaction, process features, and enzyme kinetics (4256). In the majority of known dinitrogen complexes, the metal has a low oxidation state and the auxiliary ligands are
phosphines, carbon monoxide, ammonia or hydride. Today, many dinitrogen complexes of transition metals have been
synthesized and characterized, but only a few of them undergo reduction to give ammonia instead of dinitrogen ligand being
easily displaced (40-41, 57-62). These electron-rich, low oxidation state systems, have the electrons necessary for the reduction
stored in the metal and use them when the preferential proton addition to the ligand takes place resulting in an increase of the
formal oxidation state of the metal proportionately.
Synthetic Methods
The preparation of mononuclear dinitrogen complexes can be broadly classified into four methods: (i) direct addition of
dinitrogen to a coordinatively unsaturated complex (ii) coordination of N2 by labile ligand replacement (iii) reduction of a
suitable transition metal complex in the presence of dinitrogen using an appropriate reducing agent and (iv) the conversion of
-
other coordinated nitrogen containing species (N2H2, N3 , N2O) into the dinitrogen ligand. Typical examples of these methods
are listed in Scheme 3. The synthesis of bridged binuclear complexes involves the reduction of a transition metal complex
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under dinitrogen or by the displacement of a labile ligand from a metal complex by certain mononuclear dinitrogen complexes
(Scheme 4). The reduction of metal halides under N2 and reaction of coordinated ammonia in an amine complex with nitrous
acid (HNO2) would produce bis(dinitrogen) complexes (Scheme 5). Some binary LnM(N2) type complexes have been
prepared by interaction of atomic metal vapors with dinitrogen gas at low temperature by matrix isolation technique {e.g. PtN2,
Ni(CO)3N2}.
(i) RuH2(PPh3)3 + N2
RuH2(N2)(PPh3)3
(ii) CoH3(PPh3)3 + N2
CoH(N2)(PPh3)3 + H2
(iii)
(iv)
Scheme 3. Selected examples of preparation of mononuclear dinitrogen complexes
[(NH3)5RuN2Ru(NH3)5]4+
(i) 2[Ru(NH3)5Cl]Cl2 + Zn/Hg + N2
(ii) [(H3N)5RuN2]2+ + [(H2O)Ru(NH3)5]2+
[(NH3)5Ru(N2)Ru(NH3)5]4+ + H2O
Scheme 4. Representative examples of synthesis of bridged binuclear dinitrogen complexes
(i)
where THF = Tetrahydrofuran, diphos = 1,2-bis(diphenylphosphino)ethane
(ii) [Os(NH3)5N2]2+ + HNO2
cis-[Os(NH3)4(N2)2]2+ + 2H2O
Scheme 5. Illustrative examples of chemical synthesis of bis-dinitrogen complexes
Structural Types
The nature of the metal, the nature of the coligands and dinitrogen coordination modes influence the degree of dinitrogen
activation. The diatomic dinitrogen ligand can bind to metals in various coordination modes (Scheme 6). The common
coordination modes of N2 in mononuclear dinitrogen complexes include the end-on linear (I), end-on bent (II), side-on
perpendicular (III) and bis-cis bent (IV) modes. The geometric arrangements found in binuclear dinitrogen complexes include
the bridging end-on (V), bridging bent-trans (VI), bridging side-on (VII) and those involving both bridging end-on and
terminal end-on (VIII) types. The geometric structures in polynuclear dinitrogen complexes include the bridging end-on linear,
non-planar and bridging-bent metal-N2 modes. In the majority of known dinitrogen complexes, N2 is bound in an end-on
fashion like CO in a metal carbonyl complex [e.g. IrCl(N2)(PPh3)2] rather than in side-on bonded like coordinated acetylene
although the latter types exist {e.g. [(η5-C5Me4H)2Zr]2(µ2,η2,η2-N2)}. The bridging side-on mode of dinitrogen coordination
has been determined crystallographycally in a few complexes such as [{(PhLi)3Ni}2(N2).2Et2O]2, {[(Pr2iPCH2SiMe2)2N]ZnCl}
2(µ-η2:η2-N2) and [{Ph(NaOEt2)2Ph2Ni2(N2)NaLi6(OEt)4OEt2}2] (63-69). The bridging end-on type of coordination is
observed in the ruthenium complex, {[Ru(NH3)5]2(µ-N2)}4+ . However, there are other possible bonding structures with low
energy barriers between them. The stability of these systems indicates the possibility of preparing M-N2 complexes with these
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configurations. Further, various M : N2 ratios such as 1:1, 1:2, 2:1, 2:3 and 3:2 are possible (70-71). These new structures
highlight the possibility of preparing novel dinitrogen complexes with many structural features incorporated in their design
leading to the functional product development.
Scheme 6. The principal coordination modes of dinitrogen ligand in some transition metal dinitrogen complexes
Bonding Aspects
The metal-dinitrogen bonding can be described by a combination of ligand to metal σ-bonding (N2
M σ-donation) and
metal to ligand π-backbonding (M
N2 π-acceptance) (Fig 2.). The ligand to metal σ -donor-bond involves the overlap
of 3σg orbital of dinitrogen and a vacant metal d-orbital. This σ-donation increases electron density at the metal and promotes
the formation of the acceptor-bond involving a filled metal d-orbital and a vacant antibonding 2π*g orbital of the dinitrogen.
Thus the stability of dinitrogen complexes is the result of this synergetic effect. The transition metal in low formal oxidation
state and the presence of coligands like phosphines, arenes and carbonyls favor this type of bonding. The increase in N-N bond
length indicates dinitrogen activation upon coordination. The N-N bond lengths in terminal end-on bonded dinitrogen
complexes are only slightly greater than that in the gaseous dinitrogen (109.75 pm). The dinitrogen in side-on bound
complexes is activated as indicated by the much longer N-N bond distances, although any particular pattern to signal the extent
of activation is yet to emerge. The decrease in M-N bond distances suggests the backbonding of electron density from the
metal σ orbital to π* antibonding orbitals of dinitrogen. The stronger the M-N bond, the weaker the N-N bond, making it more
reactive toward reduction. The structural features of the metalloenzyme and the N2-binding modes in the different FeMo/VFe/
FeFe cofactors during nitrogenase turn over need to be addressed. The structure-bonding relationships will have the greatest
impact in defining the reactivity at a transition metal site.
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Fig 2. Simplifiled diagram of transition metal-dinitrogen bond
Reactivity Features
There are several functional models of N2ase where coordinated dinitrogen is activated enough and consequent reduction of
activated dinitrogen occurs along with liberation of ammonia. The stable bis-dinitrogen complexes of Mo and W, [M(N2)2
(PMe2Ph)4], M = Mo or W react with acids (H2SO4 in MeOH) to yield NH3 under mild conditions (59-60). Similarly, the
protonation of N2 model system, trans-[Mo(N2)2(dppe)(PPh2Me)2 with HBr in the presence of SnCl2 yields NH3 and N2H4 as
products (72). The protonation of [{Mo(C5Me5)Me3}2N2] with HCl produces NH3 in low yield (108). It has been shown
recently that the iron(0) dinitrogen complex, [Fe(N2)(dmpe)2], dmpe = (CH3)2PCH2CH2P(CH3)2 produces NH3 when the
coordinated dinitrogen is protonated upon reaction with acids, and H2 and N2 are also evolved in the mechanistically unknown
reaction (118). Of particular importance to the reduction would be the protonation of the vanadium complex [V(C6H4CH2Me2)2
(C6H5N}2] which reacts with HCl producing NH3 and N2 as minor products. The complex [V(N2)2(dmpe)2]- reacts with HCl
to yield small quantities of ammonia. The acidic dihydrogen complex, [Ru(C5H5)(diphosphine)(η2-H2)]+ (diphosphine =
PR'2CH2CH2PR'2, R' = P-CF3C6H4) protonates coordinated dinitrogen in the complex [W(N2)2(dppe)2], dppe =
Ph2PCH2CH2PPh2, to produce the hydrazido complex [W(NNH2)(F)(dppe)2]BF4.
The study of complexes containing N2H2, N2H4 and NH3 could serve as suitable chemical models of intermediate stages for
nitrogenase action. It is necessary to increase understanding of the chemical basis of the reduction mechanism of nitrogen
fixation. The protonation of coordinated dinitrogen provides a vehicle for the study of the essential steps of the nitrogen
fixation process using the metalloenzyme. While it is important to recognize research direction and subsequent successes of the
past, the realistic model for the fundamental nitrogen fixation reaction that works under catalytic conditions remains to be
discovered. It is remarkable to note that in developing solutions to nitrogen fixation challenge we have to adopt as much
biological realism as possible, by taking into consideration the various components of the enzyme structure-function
interrelationships such as bond lengths, interbond angles, overall geometry, electron release probability and mechanistic
aspects of reduction activity. Other experimental operating conditions such as pH, temperature, catalyst concentration, nitrogen
partial pressure also play a crucial role in the design of suitable catalytic functional model. Large variation in the oxidation
state of the single metal is unlikely to be involved in the actual enzymatic process as observed in biomimetic complexes, and
the biological reducing agents are not strong enough to maintain zerovalent state of the metals involved.
Other reactions of dinitrogen complexes include the following: (i) oxidation of dinitrogen complexes with/without complete
loss of dinitrogen (ii) ligand displacement reactions with N2 evolution/replacement (iii) displacement of coligands with
retention of the metal-dinitrogen bond (iv) addition reaction to unsaturated dinitrogen complexes. Due to easy displacement of
dinitrogen certain complexes have been shown to catalyze different reaction types like isomerization, polymerization and
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hydrogenation reactions. These reactions without a specific relationship to dinitrogen activation are not discussed here and they
may have other industrial uses not yet realized (73-127). The lessons from the conceptual content and emerging trends in other
domains reveal radical ideas that should inspire process innovation in the field.
Heterogeneous Catalysis
The synthesis of ammonia by Haber process involves iron catalyst along with small amount of promoters like potassium oxide
and aluminum oxide (T = 450o C, P = 250 atm). The rate determining step in the combination of dinitrogen with dihydrogen to
produce ammonia is the dissociation of N N triple bond at high temperature. Although ammonia synthesis mechanism is not
clearly understood, the possible catalytic reduction pathway could involve the following seven functional steps (Fig3.).
1) proper approach of adsorbing species: diffusion of the dinitrogen (N2) and dihydrogen (H2), into the surface of the iron
catalyst for surface interaction
2) binding and activation: adsorption of the gaseous reactants at the active surface of the catalyst
N2(g)
N2(ads), H2(g)
H2(ads)
3) surface reactions: dissociation of the adsorbed gases at the adjacent surface
N2(ads)
2N(ads) , H2(ads)
2H(ads)
4) promotion of subsequent protonation: quick chemical interaction between the adsorbed nitrogen and hydrogen to produce
unstable intermediate compounds
N(ads) + H(ads)
NH(ads), NH(ads) + H(ads)
NH2(ads),
NH2(ads) + H
NH3(ads)
5) formation of final product: generation of ammonia as the final product through a series of insertion reactions
N(ads) + H(ads)
NH3(ads)
6) negative adsorption: desorption of the product of reduction to the gas phase from the surface of the catalyst
NH3(ads)
NH3(g)
7) product separation: diffusion of the ammonia out of the catalyst surface by having less affinity and into the bulk stream gas
surroundings and regeneration of the iron catalyst in its original state as part of the overall catalytic process.
The catalyst induces a specific chemical response that leads to the formation of a continuous interface between the catalytic
surface and substrate molecules and prevent formation of unwanted byproducts. Common criteria used to determine the
catalyst performance is by the parameters such as activity, selectivity, thermal and mechanical stability, design life and cycle
life and overall production costs. As ammonia is the single thermodynamically favorable final product in the interaction of
dinitrogen and dihydrogen, selectivity factor can be neglected. The discovery of artificial N2ase for industrial fixation with
environmentally benign technology can be of great importance specific to naturally nitrogen-poor soils to make it nitrogenrich, thereby increasing soil fertility without substantial environmental damage. The recent research trend suggests the
molecular manufacture of nanoscale catalysts that significantly enhance the chemical reactivity rate. The development of metal
cluster catalysts and smart catalysts that influence fixation rate by forming suitable bonding interaction between a catalyst site
and an adsorbed molecular species is a welcome change to refocus. The availability of high-resolution transmission electron
microscope (HRTEM) in several research laboratories makes it possible to note the surface defects that act as potential
adsorption sites for catalysis. This information can be obtained from the analysis of TEM micrograph and it is often possible to
predict the actual mechanism of catalytic reaction under consideration.
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Fig 3. Diagram of the catalytic reduction of dinitrogen on a surface showing substrate depletion or product
accumulation
Concluding Remarks
Available soil nitrogen is not sufficient for intensive crop production and more dynamic research in different directions is
needed as a step to establish forward movement to inspire and educate young minds about nitrogen fixation with several
distinct advantages. There are three major types of nitrogenases with considerable variation in product specificity, source, and
reaction conditions. To induce molecular nitrogen to react at commercially useful rates, it is usually necessary to make use of
active catalysts, to elevate the temperature, to use high pressure, or to use a catalyst in conjunction with elevated temperatures,
or to use ultrasound/hydrodynamic cavitation process to generate local hot spots. It is reasonable to expect important future
discoveries about nitrogen fixation towards its successful application to increase agricultural yields and put prominent ideas to
practical use to be of educational value based on the intense interest and research activity in the field by dedicated researchers.
It is particularly important to identify the presence of different new species of bacteria that live at subzero temperatures and
those that survive at high temperatures and to establish the gene specificity to its functionality at temperature extremes. One of
the quests for genetic modification research (GMR) is to harvest many varieties of modified nitrogen fixation plants and zero
contamination is not possible at present.
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In spite of the continued efforts, using the tools and techniques available today, the quest for an efficient synthetic model on an
industrial scale of the catalytic site of nitrogenase enzyme remains an open question with a rational, responsible approach to the
exploration. The use of X-ray crystallography is a major factor contributing to this development in its early stages. The crystal
growth techniques, X-ray data collection, structure solving and other aspects of X-ray crystallography are described in detail in
several excellent references that provide the most detailed information on enzyme structure determination (124-125). The
application of modern instrumental techniques like extended X-ray absorption fine structure spectroscopy (EXAFS) and
improved spectroscopic methods will prove to be extremely influencing factors in the years to come in attempts to address the
problem of nitrogen fixation by suitable design of new catalysts and reactions. The studies of electron transfer pathways in
nitrogen fixing systems by low temperature matrix isolation technique to identify reaction and catalytic intermediates, lowtemperature detection of transient species by nuclear magnetic resonance (NMR) spectroscopy and the detailed structural
information on N2ase enzymes provide important information for complete characterization of complex metalloproteins. To
detect nuclei coupled to the FeMoco electron-nuclear double resonance (ENDOR) spectroscopic technique is considered useful
(126).
The electronic and steric effects of catalysts for dinitrogen reduction reaction in the light of the metal-dinitrogen binding
interactions by a combination of σ-donor and π-acceptor properties have to be studied spectroscopically to find out in vitro
nitrogen fixation process working under mild conditions. It is clear that a great deal remains to be discovered and a truly
interdisciplinary and intensive effort will be required to find out a proper solution to the problem. The recent advances in BNF
indicates that chemical nitrogen fixation models need to be refined at the molecular level. Futher, extensive research is required
in metal dinitrogen chemistry by constructing complexes containing Mo, Fe, V, and S because of their presence in biological
systems. It gives the opportunity to carefully examine their chemical properties that can lead to an understanding of reactivity
features feasible in metalloenzymes under physiological conditions. There are some opportunities to achieve the following
research objectives: to create nanostructures that help in the selective absorption of dinitrogen for further transformation and
genetically engineered bacteria to develop transgenic plants for dinitrogen transformation leading to the understanding of the
complex biochemical reaction trajectories, to develop immobilized biocatalysts that have strict substrate specificity using
immobilization techniques such as physical entrapment, retention by membrane, cross linking with inert protein or chemical
bonding to a support to create multinuclear cluster type active site capable of binding and activating the dinitrogen by flexible
electron reservoirs. In addition to exploratory synthetic work on dinitrogen complexes, focus on further investigation exploiting
them to end-use is equally important. This is primarily because the design of sustainable chemical process for selective
reduction of dinitrogen is still an important goal of nitrogen fixation research for the future. The nitrogen fixation continues to
remain one of the mysteries of nature with a scope for further research on chemically appropriate procedures and quite
attractive educational topic because of invaluable learning experience in the domain.
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