nitrogen fixation and dinitrogen complexes -...

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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 e-learning 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 inter-collegiate 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 bio-resource 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 structure-function 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 Artificial Processes

1. Biological Nitrogen Fixation (BNF) Direct assimilation of atmospheric nitrogen by some micro-organisms

R.T. atm. N2 + 8H+ + 8e- 2 NH3 + H2

N2ase

Industrial Nitrogen Fixation (INF) Commercial production of ammonia by Haber-Bosch process

450° C, 250 atm N2 + 3 H2 2 NH3

Fe/K2O/Al2O3

2. Incidental Electrical Fixation Formation of oxides of nitrogen from the action of lightning in the upper atmosphere by electrical discharges

lightning N2 + O2 2 NO

2NO + O2 2NO2

Birkeland-Eyde Process Dinitrogen oxidation to generate nitric oxide by electric-arc process

3000° C N2 + O2 2 NO

Electric discharge

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

Frank-Caro Cyanamide Process Calcium carbide reacts with nitrogen to give calcium cyanamide

1100° C CaC2 + N2 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 cubane-type 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 high-pressure 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.

N2ase N2 + 16MgATP + 8e- + 8H+ 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]-, [V-Fe] - 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 (42-56). 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

(i) 2[Ru(NH3)5Cl]Cl2 + Zn/Hg + N2 [(NH3)5RuN2Ru(NH3)5]4+

(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 nitrogen-rich, 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, low-temperature 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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