Draw A Labeled Diagram Of The Nitrogen Cycle

Nitrogen Cycle

P. Cabello , ... C. Moreno-Vivián , in Encyclopedia of Microbiology (Third Edition), 2009

Ecological and Environmental Aspects of the Nitrogen Cycle

The nitrogen cycle reactions have important environmental, agronomic, and health implications and may be also used for technical applications. Thus, nitrification, denitrification, and anammox are important processes for removal of nitrogenous compounds in the treatment of industrial or domestic wastewater.

Nitrogen is usually an essential nutrient that limits plant growth. Therefore, to produce plants of quality with high yield and sufficient protein content, it is necessary to fertilize agricultural soils with mineral fertilizers (synthesized by the Haber–Bosch process, which needs high energy consumption), or manure, a mixture of both inorganic and organic nitrogenous compounds that are mineralized by soil microorganisms. The Rhizobium–legume symbiosis has great agricultural and ecological importance since it provides a source of fixed nitrogen for plant growth without the need for fertilizers. Also in rice paddies, the symbiotic N₂ fixation based on Anabaena azollae, together with rice rhizosphere-attached diazotrophic strains of Azotobacter and Azospirillum, allows rice growth without fertilizer addition.

In fertilized croplands, a certain amount of ammonium is oxidized to nitrite and nitrate, which leach easily from soils into ground and surface waters. Thus, excessive use of fertilizers leads to accumulation of nitrate and nitrite in groundwater causing environmental and health problems. The main environmental threat is the eutrophication of aquatic ecosystems by excessive growth of bacteria and algae. Consumption of drinking water with high nitrate content may causes methaemoglobinaemia and gastric cancer. Denitrification has a positive effect because it decreases nitrate leaching to groundwater, but has also negative effects since it causes important losses of nitrogen from soils and it is a source of the greenhouse gas N₂O. In agricultural soils, losses by denitrification may represent up to 30% of applied fertilizer. Nitrogen oxides formed by incomplete denitrification and nitrification have a great impact on the atmosphere chemistry. N₂O is a stable gas (over 100 years of lifetime) that shows 320-fold more greenhouse effect than CO₂, contributing to global warming. Also, in the stratosphere, NO and N₂O participate in reactions that destroy the ozone layer, which protects life against UV radiation. Atmospheric NO is also oxidized to NO₂ that after hydration generates HNO₃, causing acid rain falls. As N₂O is not harmful for organisms, the absence of Nos activity in some denitrifiers does not represent a problem for the bacteria but has serious environmental consequences. Nitrifier denitrification, mainly by marine AOB, also contributes to N₂O emissions. If denitrification is the main pathway for nitrogen losses in terrestrial ecosystems, anammox may account for up to 67% of N₂ production in marine environments. Nitrite availability likely controls the abundance of anammox bacteria, which are usually linked to the activity of other bacterial groups that produce nitrite by nitrification or denitrification.

Interactions among organisms affect significantly the nitrogen cycle processes. N₂ fixation in natural ecosystems is greatly affected by associative bacteria that potentially benefice the plants. More than 80% of higher plants are colonized by arbuscular mycorrhizal fungi, which interact with diazotrophic bacteria and assimilate nitrate and ammonium that are finally transferred to the plant. Another special interaction among organisms occurs in the gut of earthworms, an anoxic habitat for denitrifying soil bacteria. Understanding of the role of microbial consortia in nitrogen cycle was limited by the fact that only 1% of the bacteria present in an ecosystem may be cultured by traditional microbiology techniques. However, modern molecular tools and environmental genomics (metagenomic) are changing our knowledge of these communities in natural habitats and their effects on the nitrogen cycle. Metagenomic studies have revealed several networks of bacterial populations catalyzing different steps of the nitrogen cycle in natural environments. Thus, although no known methanotroph is able to denitrify, various microorganism consortia may use methane as carbon source to carry out denitrification aerobically or anaerobically. Aerobic methane oxidation coupled to denitrification is performed by aerobic methanotrophs that release soluble organic compounds (methanol) that are used as electron donor by denitrifiers. A bacterium–archaeon consortium also couples denitrification to methane oxidation under anaerobiosis, although this microbial consortium grows very slowly and its potential contribution to the global nitrogen cycle is unknown.

The nitrogen cycle is profoundly affected by anthropogenic impacts, like the high input of nitrogen to the biosphere through application of synthetic fertilizers, use of N₂-fixing crops in agriculture, release of nitrogen oxides by combustions, and industrial production of different nitrogenous compounds that pollute worldwide regions. This input of human-produced nitrogen has greatly stimulated nitrifier denitrification, with concomitant increases in N₂O emissions. Mitigation of imbalances in nitrogen cycle will be required to reduce microbial production of greenhouse gases and global warming.

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Research on Nitrification and Related Processes, Part A

Melanie Kern , Jörg Simon , in Methods in Enzymology, 2011

Abstract

Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c.

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NUTRITION | Nitrogen Fixation

K. Fisher , W.E. Newton , in Encyclopedia of Applied Plant Sciences, 2003

Biological Nitrogen Cycle

The nitrogen cycle describes the major sinks, nitrogen-transfer pathways, and transformations involved in the global cycling of nitrogen ( Figure 1). Atmospheric N₂ is 'fixed' by both biological and nonbiological processes and results in formation of either reduced or oxidized compounds of nitrogen. These nitrogen compounds are added to the soil and become available for assimilation by bacteria and plants. They return the fixed nitrogen to the soil when they die and decay. Other soil bacteria convert ammonia into nitrate and then nitrate back to N₂ to complete the cycling of nitrogen. These latter processes lead to a loss of usable fixed nitrogen from the soil. Overall, the input rate from biological, spontaneous (e.g., lightning), and industrial nitrogen fertilizer (by the Haber–Bosch process) production is slightly higher than the loss rate. Of these, the major input of fixed nitrogen is through biological nitrogen fixation by microorganisms known collectively as diazotrophs. The nitrogenase in these important organisms operates at ambient temperature and pressure in the soil with the sun as the ultimate energy source. N₂-fixing organisms have little in common with each other apart from being prokaryotic and diazotrophic. They are generally classified into two main groups; those that are free-living, and those that are symbiotic, i.e., that fix N₂ in association with a plant, usually with its root. Free-living bacteria may be either phototrophic or chemotrophic and either heterotrophic or autotrophic. Moreover, they may fix N₂ under anaerobic, microaerobic, or aerobic conditions.

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Research on Nitrification and Related Processes, Part B

Willm Martens-Habbena , David A. Stahl , in Methods in Enzymology, 2011

1 Introduction

The nitrogen cycle has changed significantly within the past two decades. New processes and microorganisms have been discovered that contribute to this still insufficiently resolved nutrient cycle ( Francis et al., 2007; Prosser and Nicol, 2008). Among the most important findings was the discovery of aerobic ammonia oxidation within the domain Archaea. Ammonia oxidation, the first step of nitrification, has been known for over a century and it was thought to be restricted to three genera of Beta- and Gammaproteobacteria (Koops et al., 2000; Kowalchuk and Stephen, 2001). Putative ammonia monooxygenase (AMO) genes linked to archaeal ribosomal RNA genes were then found on fosmid clones from soil, suggesting a possible role of mesophilic archaea in the nitrogen cycle (Schleper et al., 2005; Treusch et al., 2005). This hypothesis was confirmed by the isolation of the ammonia-oxidizing archaea ammonia-oxidizing archaeon (AOA) Nitrosopumilus maritimus strain SCM1 (Könneke et al., 2005), and the description of additional mesophilic and thermophilic archaeal enrichment cultures that stoichiometrically oxidize ammonia to nitrite (de la Torre et al., 2008; Hatzenpichler et al., 2008; Schleper and Nicol, 2010; Wuchter et al., 2006).

Detailed molecular surveys demonstrated that AOA are ubiquitous in marine and terrestrial environments and frequently outnumber AOB, especially in nutrient-poor environments (Beman et al., 2010; Herrmann et al., 2009; Leininger et al., 2006; Mincer et al., 2007; Prosser and Nicol, 2008; Zhang et al., 2010). AOA account for up to 30% of the microbial plankton in the oligotrophic ocean gyres (Agogué et al., 2008; Karner et al., 2001) and between 1% and 3% of the total microbial count in soils and sediments, together indicating that these poorly understood microorganisms belong to the most abundant microbial clades on Earth (Karner et al., 2001; Prosser and Nicol, 2008).

Metagenomic studies on marine and soil fosmid clones, the uncultured sponge-associated archaeon, Cand. Cenarchaeum symbiosum, and the genome sequence of N. maritimus have offered new insights into the metabolism and phylogeny of these organisms (Brochier-Armanet et al., 2008; Hallam et al., 2006a,b; Spang et al., 2010; Treusch et al., 2005; Walker et al., 2010). Phylogenetic studies on ribosomal proteins and core genes indicated that AOA represent a novel phylum, Thaumarchaeota, within the Archaea (Brochier-Armanet et al., 2008; Spang et al., 2010). Thus far, ammonia oxidation is the only plausible pathway of generation of metabolic energy by these organisms, although the investigated (meta-) genomes do not share the ammonia oxidation pathway found in AOB. AOB possess an AMO, which derives electrons from the ubiquinone pool to oxidize ammonia to hydroxylamine. Hydroxylamine is subsequently oxidized to nitrite by a hydroxylamine oxidoreductase (HAO), which delivers electrons back into the ubiquinone pool for respiration and further activation of AMO (Arp et al., 2007; Hooper, 1989; Walker et al., 2010). Whereas a canonical AMO is consistently found in AOA (meta-) genomes, a putative HAO has not been identified (Hallam et al., 2006a,b; Treusch et al., 2005; Walker et al., 2010). Thus, energy generation in these archaea likely follows a novel pathway. Following the most parsimonious hypothesis, the archaeal AMO oxidizes ammonia to hydroxylamine similar to the bacterial pathway. Hydroxylamine would subsequently be oxidized to nitrite by one or multiple novel enzymes, for example, putative multicopper oxidases, which could function analogously to the bacterial HAO. Alternatively, the archaeal AMO could yield a more oxidized product than hydroxylamine (e.g., H₂N₂O₂ or HNO), that may subsequently be oxidized to nitrite via one of the putative multicopper oxidases, and channel electrons via plasto- and sulfocyanins into the mostly Cu-based respiratory chain typically found in AOA (meta-)genomes (Schleper and Nicol, 2010; Walker et al., 2010).

Along with a different ammonia oxidation pathway and greater involvement of Cu-dependent enzymes, AOA possess strikingly different cellular characteristics than the AOB. The size of N. maritimus cells and their genome is close to the estimated lower limits of free-living organisms (Button, 2000) and comparable to those of Pelagibacter ubique (Rappé et al., 2002). Similar to P. ubique, SCM1 cells are only between 0.5–0.9 μm long and 0.25 μm wide and contain approximately 10 fg protein (≈ 16–20 fg dry weight cell per cell) (Martens-Habbena et al., 2009). Thus, even a single copy of the 1.645 Mbp Nitrosopumilus genome accounts for almost 10% of cellular dry weight. In contrast, cells of AOB strains in culture have at least 10-fold higher biomass per cell, equivalent to between 120 and up to 1000 fg protein per cell (Keen and Prosser, 1987; Martens-Habbena et al., 2009), and their genome size ranges from 2.8 to 3.5 Mbp (Arp et al., 2007). Small cell size, and associated increase in surface-to-volume ratio, as well as small genome size have previously been considered important evolutionary adaptations by oligotrophic microorganisms to life under resource limitation in nutrient-depleted environments (Button, 2000; Giovannoni et al., 2005; Harder and Dijkhuizen, 1983; Roszak and Colwell, 1987). In conjunction with the abundance patterns revealed by molecular surveys, these distinct characteristics suggested that AOA and AOB could occupy different ecological niches and that AOA could be particularly adapted to live in oligotrophic environments (Martens-Habbena et al., 2009).

Under nutrient-limited conditions, growth and survival of microorganisms depend critically on their ability to scavenge nutrients from the surrounding environment, reflected by kinetics of nutrient uptake and energy source oxidation (Button, 1985; Button et al., 2004). We therefore sought to determine and compare the kinetic characteristics of ammonia uptake and oxidation of N. maritimus and known AOB strains. Slow growth rates and low biomass yields obtained in laboratory cultures of AOB and especially of N. maritimus make such kinetic studies and physiological experiments in general difficult. Further, growth of N. maritimus is strongly impaired by agitation, rendering nutrient-limited chemostat experiments challenging. We therefore elected to use microrespirometry to determine the stoichiometry and kinetics of ammonia oxidation via its associated oxygen consumption.

In this chapter, we describe this microrespirometry setup and review kinetic experiments conducted with N. maritimus, as well as ammonia- and nitrite-oxidizing bacteria. The setup was particularly tuned to monitor activity of microorganisms in very dilute cultures. The high resolution and low detection limit of approximately 0.2 μM O₂ uptake per hour permitted us to continuously monitor ammonia oxidation activity in undisturbed N. maritimus cultures without harvesting to concentrate cell material. Using this technique, we were able to accurately determine the stoichiometry and kinetics of ammonia oxidation by N. maritimus, demonstrating that this organism has one of the highest substrate affinities found among microorganisms. This physiology strongly supports the hypothesis that N. maritimus is among the most oligotrophic organisms known to date. Altogether, the available data indicate that oligophilic AOA may have significant impact on the nitrogen and carbon cycles of the global oceans (Martens-Habbena et al., 2009).

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Research on Nitrification and Related Processes, Part B

James E. Graham , ... Martin G. Klotz , in Methods in Enzymology, 2011

3.1 N-cycle organisms and reactive nitrogen transformation pathways

The nitrogen cycle includes a wide array of intermediates that are classified as either fixed (solid or liquid) or volatile (gaseous), or as inert (N ₂) or reactive (all other intermediates). The major fixed reactive nitrogen species include ammonium (NH₄ ⁺), nitrite (NO₂ ⁻), and nitrate (NO₃ ⁻) and their interconversion through intermediates such as hydroxylamine (NH₂OH) or nitroxyl (nitrosyl hydride, HNO) and their removal via fixed (hydrazine, N₂H₄) or volatile (nitric oxide, NO; nitrous oxide, N₂O) intermediates to dinitrogen (N₂) is facilitated by a diverse set of molecular inventory employed by numerous microorganisms. Since the interconversions represent redox processes, most of them are either coupled to dissimilatory catabolic electron flow tied to oxidative phosphorylation and anaerobic respiration or part of anabolic electron sink reactions during nitrogen assimilation (see Fig. 14.2).

Dissimilatory nitrate reduction to dinitrogen, the major microbial nitrogen removal process in marine ecosystems, occurs in form of two major pathways under hypoxic and anoxic conditions, respectively: classical denitrification with nitrous oxide as a mandatory intermediate and denitrifying ammonia oxidation also known as anammox (Fig. 14.2; Klotz and Stein, 2010). In classical denitrification, the transformation of nitrate to nitrite to nitric oxide to nitrous oxide and often also to dinitrogen, nitrite reduction to nitric oxide is the rate-limiting step that is catalyzed by either copper-containing NirK or cytochrome cd-1 NirS nitrite reductase enzymes (Zumft, 1997; Zumft and Kroneck, 2006). The genes encoding their catalytic subunits (nirK and nirS) have been used extensively as targets to study the structure of nitrite reducer communities in aquatic and terrestrial environments, and these studies were often amended with probing for genes encoding nitric oxide (i.e., norB, norZ) and/or nitrous oxide (nosZ, nosW) reductases to identify the classical denitrifier community (Avrahami et al., 2002; Braker and Tiedje, 2003; Braker et al., 1998, 2000, 2001; Falk et al., 2010; Oakley et al., 2007; Prieme et al., 2002; Santoro et al., 2006; Simon et al., 2004). Classical denitrification constitutes one of the main forms of anaerobic respiration and is performed by a great diversity of heterotrophic bacteria including many pathogens of humans and other animals.

Classical denitrification has long been considered the only pathway for nitrogen loss from marine and terrestrial ecosystems (Zumft, 1997; Zumft and Kroneck, 2006). However, since 1999 evidence for denitrification during anaerobic oxidization of ammonia (anammox) as another major pathway for nitrogen removal has emerged (Fig. 14.2; Dalsgaard et al., 2005; Francis et al., 2007; Jetten et al., 2005; Kuenen, 2008; Schmidt et al., 2003; Strous et al., 1999). NO-forming nitrite reductase-encoding genes have been identified in the genomes of anammox bacteria; however, nitrous oxide-forming nitric oxide reductase genes are absent from anammox bacterial genomes (Jetten et al., 2009; Strous et al., 2006). Because nitrite-derived nitric oxide is the sole oxidant of ammonia in the anammox process (Jetten et al., 2009), both divergent denitrification pathways compete fiercely for nitrite (Kartal et al., 2007). Likewise, the molecular inventory responsible for denitrifying N-removal competes for the same nitrite pool with the inventory involved in ammonification, which retains the transformed nitrogen in the system. Both assimilatory nitrate reduction to ammonia (ANRA) and dissimilatory (respiratory) nitrate reduction to ammonia (DNRA) interchangeably employ one of the molybdopterine guanine dinucleotide-based enzymes for nitrate reduction: periplasmic dissimilatory (napA) and the cytoplasmic soluble (nasA) and membrane-bound respiratory (narG) nitrate reductases; any of which may also participate in the nitrate reduction step of denitrification (Fig. 14.2). Detection of nitrate reduction inventory per se is thus not useful for identifying or discriminating between either form of denitrification as well as ammonification. On the other hand, ammonifiers use dedicated sets of enzymes and reductant shuttles for the reduction of nitrite to ammonia. For ANRA, microbes employ assimilatory cytoplasmic ferredoxin-dependent (the nirA gene product in Cyanobacteria and Epsilonproteobacteria) or NADH-dependent (the nirB/nasB gene product in Beta- and Gammaproteobacteria) nitrite reductases, both of which contain a single siroheme and a [4Fe–4S] center (Malm et al., 2009; Martiny et al., 2009; Moreno-Vivian et al., 1999). For DNRA, the major enzyme is the respiratory pentaheme cytochrome c nitrite reductase (nrfA; Pittman et al., 2007; Simon, 2002; Smith et al., 2007). A novel dual N assimilation and respiratory mechanism employing the reverse hydroxylamine-ubiquinone redox module (HURM; Klotz and Stein, 2008) pathway (haoA'+ cycB) has been reported recently (Campbell et al., 2009). Interestingly, the nrfAH and haoA'+cycB inventories are homologues (Bergman et al., 2005; Kim et al., 2008; Klotz et al., 2008).

Ammonia, whether available in the environment, obtained by nitrogen fixation or by ammonification from NOx, is another important pool of reactive nitrogen. When not assimilated into biomass by respective pathways employing glutamate dehydrogenase, GS-GOGAT or alanine dehydrogenase or removed from the system by the anammox process, the major transformation pathway is nitrification (Fig. 14.2). Nitrification is defined as the aerobic oxidation of ammonia to nitrite followed by the aerobic oxidation of nitrite to nitrate. Together with DNRA, ANRA, assimilatory, and respiratory ammonification, nitrification represents one of the key transformation processes between different fixed nitrogen intermediates (Fig. 14.2; Allen et al., 2001; Brandes et al., 2007; Butler and Richardson, 2005; Ferguson and Richardson, 2005; Jepson et al., 2006; Klotz and Stein, 2008; Lin and Stewart, 1998; Moreno-Vivian et al., 1999; Potter et al., 2001; Simon, 2002; Smith et al., 2007; Tavares et al., 2006; and references therein). Although nitrifying bacteria and the nitrification process have been studied for more than 100 years (Arp and Bottomley, 2006; Bock et al., 1991; Prosser, 1989; Winogradsky, 1892), our knowledge of the molecular underpinnings of both was restricted to sequences of genes encoding rRNA and the key enzymes involved in nitrogen transformations; this has changed dramatically the genomic era (Arp et al., 2007; Klotz and Stein, 2010; and references therein). In addition, the discovery of broadly distributed ammonia-oxidizing archaea (de la Torre et al., 2008; Hallam et al., 2006; Hatzenpichler et al., 2008; Könneke et al., 2005; Leininger et al., 2006; Martens-Habbena et al., 2009; Nicol and Schleper, 2006; Prosser and Nicol, 2008; Walker et al., 2010) and cohorts of taxonomically diverse methanotrophic bacteria with the ability to nitrify (Nyerges and Stein, 2009; Poret-Peterson et al., 2008) has extended the list of nitrifying microorganisms significantly. While the ammonia-oxidizing archaea also aerobically denitrify without a nitrous oxide intermediate (Bartossek et al., 2010; Klotz and Stein, 2010; Schleper and Nicol, 2010; Walker et al., 2010), AOB and nitrifying methanotrophs are also capable of (aerobic) nitrifier denitrification (Sutka et al., 2003, 2006; Wrage et al., 2001, 2004) because they produce and release NO and N₂O in the presence of oxygen. The recently described anaerobic methane-oxidizing bacterium, Methylomirabilis oxyfera in the deep-branching phylum NC10, also has the genetic potential to nitrify (oxidize ammonia to nitrite via hydroxylamine using particulate methane monooxygenase and hydroxylamine oxidoreductase) in an anoxic environment by using the dioxygen produced by dismutation of NO (Ettwig et al., 2010). Because the dismutation of NO also produces dinitrogen (Ettwig et al., 2010), the anaerobic oxidation methane by M. oxyfera is thus coupled to denitrification (Ettwig et al., 2008, 2009) without a nitrous oxide intermediate (Ettwig et al., 2010). It thus appears that M. oxyfera harbors the necessary inventory to facilitate a closed nitrogen cycle within the cell (nitrification, ammonification, denitrification), which may allow this bacterium to thrive in environments with varying external ammonia and nitrite concentrations. The regulation of expression of relevant genetic inventory is likely very complex and presents itself as a likely target for testing hypotheses by applying the described methodology for the detection and quantification of steady-state mRNA levels using RT-qPCR.

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Research on Nitrification and Related Processes, Part B

Oliver Einsle , in Methods in Enzymology, 2011

1 Introduction

Within the biogeochemical nitrogen cycle, the most oxidized form of nitrogen, nitrate (NO ₃ ⁻), and the most reduced one, ammonium (NH₄ ⁺), are linked through the anaerobic respiratory pathway of dissimilatory nitrate ammonification (Berks et al., 1995; Einsle and Kroneck, 2004; Richardson et al., 1998; Simon, 2002). In only two steps, nitrate is first converted to nitrite (NO₂ ⁻) by a molybdenum-containing nitrate reductase and subsequently reduced by six electrons to ammonium by the pentaheme cytochrome c nitrite reductase, NrfA (Cole, 1982; Einsle, 2001; Kaije and Anraku, 1986; Liu and Peck, 1981; Schumacher and Kroneck, 1991b). Electrons for this reaction are commonly obtained through the oxidation of formate (nitrite reduction with formate, nrf) (Motteram et al., 1981), and catalysis proceeds according to

(16.1) ${NO}_{2}^{-} + 6 e^{-} + 8 H^{+} ⇆ {NH}_{4}^{+} + 2 H_{2} O$

Microorganisms utilize this pathway to generate a proton motive force by a Q-loop mechanism involving membrane-bound formate dehydrogenase and a dedicated quinol dehydrogenase that forms part of the Nrf system. Different types of quinol-oxidizing systems have been described in context with NrfA enzymes (Fig. 16.1). When NrfA was first described in γ-proteobacteria such as Escherichia coli (Cole, 1968; Fujita and Sato, 1966), its gene was found in a genetic context of the type nrfABCDEFG, with nrfA coding for the actual enzyme, nrfB coding for a small, pentaheme electron transfer protein, nrfC and nrfD for the membrane-integral quinol dehydrogenase, and nrfE, nrfF, and nrfG for components of a dedicated assembly machinery required for attachment of the active site heme group (Eaves et al., 1998) (see below). In δ- and ε-proteobacteria and most other families of eubacteria, the nrf operon contains a gene for a membrane-integral tetraheme cytochrome c of the NapC/NirT family designated nrfH (Simon et al., 2000). In these organisms, NrfA and NrfH form a membrane-associated respiratory complex on the extracellular side of the cytoplasmic membrane that optimizes electron transfer efficiency to the terminal acceptor nitrite (Simon et al., 2001).

Besides its function in energy metabolism, NrfA might also play a role in providing reduced nitrogen for the cell (Cole, 1982). It generates ammonium in the periplasm that can subsequently be imported into the cytoplasm via ammonium transporters of the Amt/Rh family to be assimilated into glutamine by cytoplasmic glutamine synthetase (Andrade and Einsle, 2007). Other roles for the enzyme were suggested to be in detoxification of nitrite in sulfate-reducing bacteria (Greene et al., 2003; Pereira et al., 2000) or as actual sulfite reductases, as discussed below.

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Mineralization and Assimilation in Aquatic, Sediment, and Wetland Systems

Patricia M. Glibert , Douglas G. Capone , in Nitrogen Isotope Techniques, 1993

I. INTRODUCTION

Knowledge of the nitrogen cycle in marine and fresh waters has been advanced tremendously by application of tracer techniques, especially those employing ¹⁵N. Why use tracer techniques? Most importantly, although changes in the concentration of a particular nutrient with time can provide information on the net balance of production or use of that nutrient, such changes are often the result of multiple metabolic reactions. Tracer procedures allow us to dissect the individual processes contributing to the net flux. Additionally, in oceanic and oligotrophic freshwater systems, the concentrations of nitrogenous nutrients often border on the analytical limit of detection for wet chemistry and, for all practical purposes, remain quite constant. Thus, concentrations alone give no information about the rates of uptake and regeneration when they are in close balance. Tracer and isotope dilution techniques allow us to discern otherwise imperceptible rates of uptake and regeneration of nitrogen.

In general, a nitrogen tracer experiment begins with the addition of a known quantity of a ¹⁵N isotopically labeled compound. The movement of that tracer into different particulate or dissolved pools is monitored with time. Tracer movement is monitored by the measurement of the change in isotopic enrichment (ratio of ¹⁵N/¹⁴N) in the substrate and particulate fractions. Thus, for example, in ¹⁵N uptake studies, one measures the progressive increase in isotopic enrichment of the particulate fraction as a function of time as the substrate is utilized. In isotope dilution studies, one initially enriches a dissolved nitrogen pool and monitors the progressive decrease in enrichment with time due to release of the unlabeled form of that nitrogen compound. One can also label the dissolved fraction with a ¹⁵N-labeled nitrogen compound and monitor the production of another labeled dissolved nitrogen product. Such would be the case in nitrification studies, where one adds labeled NH₄ ⁺ and follows the production of ¹⁵N-labeled NO₃ ⁻. The use of ¹⁵N in measuring these varied pathways is shown in Fig. 1.

In the past decade, there has been a proliferation of measurements taking advantage of nitrogen tracer techniques. These techniques have allowed the estimation of the rates of uptake and transformation of all the major organic and inorganic species of nitrogen in a broad range of fresh- and seawater systems. They have yielded estimates of mineralization, regeneration, and nitrification in water and sediments. Isotopic procedures have been used to determine the physiological preferences of phytoplankton for different forms of nitrogen and to assess the nutritional status of phytoplankton. With increasing sophistication of these methods has come a greater awareness for the problems and pitfalls of the techniques. Early measurements using ¹⁵N in uptake experiments were, from our current perspective, very simplistically applied. For example, there was little recognition that concurrent processes, such as regeneration, could compromise a tracer uptake measurement. Our goal in this chapter is to review methods for determination of uptake and regeneration (release and mobilization) of the major forms of nitrogen in the water column and sediments. While we emphasize the problems and pitfalls, we do not intend to discourage the use of these tools but, rather, aim to provide a rigorous foundation for their application. No other current techniques can yield comparable data on nitrogen flux. Other recent reviews of ¹⁵N methodology and applications in coastal and marine waters (Harrison, 1983; Dugdale and Wilkerson, 1986; Glibert, 1988; Paasche, 1988) deal with some additional aspects of the interpretation of these types of experiments.

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Molybdenum, Tungsten, Vanadium, and Chromium

Robert R. Crichton , in Biological Inorganic Chemistry (Second Edition), 2012

Nitrogenases

In the biological nitrogen cycle ( Chapter 18), an important role is played by a relatively limited number of anaerobic microorganisms capable of converting about one-third of atmospheric dinitrogen into ammonia, which can subsequently be incorporated into glutamate and glutamine, and from there into other nitrogen containing molecules. This represents about 10⁸ tons/year, about the same as is produced by the Haber–Bosch industrial process – albeit that the latter functions at both high pressures (150–350 atm) and high temperatures (350–550 °C). The microorganisms which fix nitrogen include the bacterium Rhizobium, involved in the symbiotic fixation of nitrogen in the root nodules of leguminous plants. Since this enzyme is extremely sensitive to oxygen, ³ the plant roots produce a haemoglobin with a high affinity for oxygen, leghaemoglobin (like haemoglobins from insect larvae or lamprey, it has the classic 'globin fold' found in mammalian haemoglobins and myoglobins – see Chapter 3), which maintains an anerobic environment around the enzyme (Downie, 2005).

All nitrogenases consist of two types of subunit, one of which contains a special Fe–S cluster, known as the P-cluster, and a second, which contains an iron and sulfur-containing cofactor which includes a different metal. This metal is usually Mo, hence, the cofactor is known as FeMoCo. However in some species, and under conditions of particular metal bio-availability, Mo can be replaced by V or even by Fe. When Mo levels are low and V is available, these "alternative" nitrogenases contain V. When both Mo and V levels are low, a third nitrogenase is produced, which contains only Fe. However, by far the greatest advances in our understanding of the structure and mechanism of nitrogenases have come from studies on the MoFe-nitrogenases from free living nitrogen-fixing bacteria like Azotobacter, Clostridium, and Klebsiella.

The overall reaction catalysed by nitrogenase is

$N_{2} {+ 8 H}^{+} + 8 e^{-} {+ 16 ATP + 16 H}_{2} O \to {2 NH}_{3} {+ H}_{2} {+ 16 ADP + 16 P}_{i}$

As we can see, the process of nitrogen fixation is extremely energy intensive, requiring both large amounts of ATP and of reducing equivalents. The nitrogenase is made up of two proteins (Figure 17.11), termed the MoFe protein and the Fe protein. The α₂β₂ heterotetrameric MoFe protein contains both the FeMo-cofactor and the P-cluster, with the functional unit constituted by an αβ dimer, containing one FeMo-cofactor and one P-cluster. In contrast, the Fe protein is a homodimer, which binds a single [4Fe–4S] cluster at the interface between the two subunits. Unlike many other multiple electron transfer reactions in biochemistry, each individual electron transfer between the Fe-protein and the MoFe protein requires the binding and hydrolysis of at least two ATP molecules. The catalytic cycles of the Fe-protein and the MoFe protein are presented in Figure 17.12. In the first of the three-state cycle of the Fe protein, the reduced Fe protein ([4Fe–4S]¹⁺), with two MgATP molecules bound, associates transiently with the MoFe protein. The two MgATP molecules are hydrolysed and a single electron is transferred from the Fe protein [4Fe–4S] cluster to the MoFe protein. The oxidised Fe protein ([4Fe–4S]²⁺) with two bound MgADP molecules then dissociates from the MoFe protein. This is the overall rate-limiting step for nitrogenase catalysis. The released Fe protein is then regenerated in two steps. The MgADP molecules are replaced by MgATP, and the [4Fe–4S]²⁺ cluster is reduced back to the 1+ oxidation state. Repetition of this cycle of association, reduction, ATP hydrolysis, and dissociation transfers one electron at a time to the MoFe protein. In the eight-state MoFe protein cycle, the MoFe protein is reduced successively by one electron, with the eight states represented by E_n. Usually, when 8 reducing equivalents have been accumulated, and 16 molecules of ATP hydrolysed, the enzyme can bind and reduce the very stable triple bond of a dinitrogen molecule to two molecules of ammonia. Concomitantly, two protons and two electrons are converted to gaseous hydrogen. Electrons derived from photosynthesis or from the mitochondrial electron transport chain are transferred to the Fe-protein.

The structure of both the MoFe-cofactor and of the P-cluster became apparent when the structures of nitrogenases were determined by high-resolution X-ray crystallography. The MoFe-cofactor (Figure 17.13a) consists of a [4Fe–3S] cluster connected to a [3Fe–Mo–3S] cluster by a previously undetected central atom (possibly a nitrogen) at one corner and three bridging inorganic sulfides. (R)-homocitrate is coordinated to the Mo atom through its 2-hydroxy and 2-carboxyl groups. The MoFe-cofactor is linked to the protein by only two residues, Cys α273 and His α442, which coordinate Fe1 and the Mo atom respectively, at opposite ends of the extended cluster. This is in marked contrast to other iron–sulfur clusters, which typically have one protein side-chain ligand per metal ion. In order to complete the coordination sphere of the eight metal centres, there are a number of additional inorganic sulfides together with bidentate coordination of the Mo atom to a molecule of homocitrate, ⁴ completing its octahedral coordination.

In the dithionite-reduced state (Figure 17.13(b)) P^N, the P-cluster can be considered as two [4Fe–3S] clusters bridged by a hexacoordinate sulfur. In the P^Ox state, which is oxidised by two electrons relative to P^N, two of the iron atoms Fe5 and Fe6 have moved away from the central sulfur atom, and are now coordinated by the amide nitrogen of Cys α87 and the hydroxyl of Ser α186, maintaining the irons in a four-coordinate state.

The Fe-protein has the protein fold and nucleotide-binding domain of the G protein family of nucleotide-dependent switch proteins, which are able to change their conformation dependent on whether a nucleoside diphosphate (like GDP or ADP) is bound instead of the corresponding triphosphate (GTP or ATP). However, nucleotide analogues which induce the conformational switch of the Fe-protein do not allow substrate reduction by the MoFe protein, nor does reduction of the MoFe protein by other electron transfer reagents (whether small proteins or redox dyes) drive substrate reduction. Only the Fe-protein can reduce the MoFe protein to a level that allows it to reduce substrates like nitrogen.

Electrons arriving at the Fe-protein are transferred to the P-cluster and from there to the MoFe protein, which is the site of interaction with dinitrogen or any of the other subrates which are reduced by nitrogenase. The redox chemistry of nitrogen reduction, on the basis of model reactions first proposed by Chatt, involves nitrogenous species at the level of diazene (N₂H₂) and hydrazine (N₂H₄) before the final release of two molecules of ammonia. Recent evidence for a diazene-derived species bound to the FeMo-cofactor supports this view, as does evidence that hydrazine (N₂H₄) is a substrate for nitrogenase. A binding site for N₂ and for alkyne substrates has been localised on the iron–sulfur face of the FeMo-cofactor defined by the Fe atoms 2, 3, 6, and 7 (Figure 17.13(c)), and ENDOR spectroscopy has shown that the alkene product of alkyne reduction is probably bound end-on to a single Fe atom of the FeMo-cofactor.

A starting point for the nitrogenase reaction pathway can be proposed from the mechanisms of N₂ reduction catalysed by organometallic complexes. A series of model studies initiated in the early 1960s by the groups of Chatt and Hidai (Chatt, 1978; Hidai, 1999) demonstrated that dinitrogen could be bound and reduced to ammonia at a single metal centre by Mo and W complexes. However, although examples of virtually all of the proposed intermediates in a "Chatt" cycle were isolated, no catalytic reduction of N₂ to NH₃ was ever achieved. Catalytic reduction of dinitrogen to ammonia at a single molybdenum centre has now been achieved by the group of Richard R. Schrock ⁵ using the HITP [3,5-(2,4,6-i-Pr₃C₆H₂)₂C₆H₃] ligand (Yandulov and Schrock, 2003; Schrock, 2005). The essential intermediates in the Chatt mechanism for N₂ reduction on a mononuclear Mo metal complex as elaborated with the recent observation of catalytic reduction by Mo complexes by the Schrock group are shown in Figure 17.14 (left). N₂ is bound to Mo (represented as M) followed by stepwise reduction and proton addition, with each intermediate remaining bound to the metal. N₂ is successively hydrogenated at a single ('distal') N until the N–N bond is cleaved after the addition of 3 e⁻/H⁺, with release of the first ammonia. The second ammonia is released following further reduction of the bound nitrido species by 3 e⁻/H⁺. This has been denoted the distal (D) pathway because as drawn, the distal N atom is protonated first and also released first as NH₃. In an alternative reaction pathway for nitrogenase (Figure 17.14, right), the two N atoms are reduced alternately, with cleavage of the N–N bond occurring only later in the reaction. While both pathways involve the stepwise reduction of the N₂ bound to a metal, the alternating one provides for the addition of protons to both N atoms in turn, delaying cleavage of the N–N bound and release of the first ammonia molecule until after the addition of 5 e⁻/H⁺.

The catalytic reduction of the dinitrogen triple bond by single-site metal nitrogen intermediates raises the question of why Nature goes to the trouble of using the complex 7Fe:9S:Mo:homocitrate cluster of the FeMo-cofactor in biological nitrogen fixation. One might have expected that a simpler one- or two-metal centre for nitrogen fixation would have been dominant in evolution if it had been biologically functional. Yet, over more than a billion years, evolutionary pressures have retained this complex cofactor-based nitrogenase system, despite the requirement for the unusual metabolite, homocitrate, and at least twenty additional proteins for its assembly and insertion. Indeed, even the "alternative" nitrogenases are thought to be minor variations on the cofactor, with V or Fe replacing Mo. As Howard and Rees (2006) pointed out at the end of their overview of biological nitrogen fixation, entitled "How many metals does it take to fix N₂?", (the number of metal atoms required is 20, corresponding to the metal composition of the FeMo-cofactor, the P-cluster, and the Fe protein) – they all seem to be required, and to date no one has found a way to simplify the system. Perhaps, after all, this simply underlines the Jeremy Knowles affirmation ⁶ 'enzyme catalysis – not different, just better!'.

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NITROGEN

G. Hanrahan , G. Chan , in Encyclopedia of Analytical Science (Second Edition), 2005

Global Outlook

As discussed, the global nitrogen cycle comprises many important chemical, biochemical, geochemical, and biogeochemical processes. Nitrogen is also tightly coupled with other elements (carbon, phosphorus, sulfur, and trace metals) and understanding these relationships will help in determining the role of living matter in biogeochemical cycles. The effect of human activity on the global nitrogen cycle is also of interest. The realization that nitrogen is commonly a limiting nutrient in plant growth has led to the invention and large-scale usage of nitrogen fertilizers, which in turn accounts for more than half of the human perturbation to the global nitrogen cycle. In addition, the widespread use of fossil fuels has led to the increased production of nitrogen oxides, which ultimately contribute to photochemical smog and acid precipitation. However, it is difficult to assess the true impact of these inputs given the extent and magnitude of natural fluxes. Only through continued study of the important processes that occur in the nitrogen cycle, will we truly understand the relative impacts.

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Nitrate

Fujio Egami , Shigehiko Taniguchi , in Methods of Enzymatic Analysis (Second Edition), Volume 4, 1974

Publisher Summary

Nitrates are components of the nitrogen cycle; nitrates have a stable nitrogen atom of the highest oxidation state. Nitrates are widely distributed throughout the biosphere. They are also found in heterotrophic organisms such as in human urine and in horse serum. The existing color reactions for nitrate are less sensitive and less specific than the determination of nitrite via diazo compounds. Therefore, methods have been developed in which nitrate is converted with a suitable reducing agent to nitrite and the nitrite is measured. However, the reducing agents so far examined are not sufficiently specific, and it is normally difficult to reduce nitrate to nitrite quantitatively. This chapter describes a method for the determination of nitrates in which a specific particle-bound formate-nitrate reductase (FNR) obtained from certain strains of Escherichia coli that have no formate-nitrite reductase activity. FNR reduces nitrate quantitatively to nitrite with formate as the specific hydrogen donor but does not cause any further reduction. FMN is added as the hydrogen carrier.

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