Nitrogen is an essential component of all living organisms and the main nutrient limiting life on our planet By far, the largest inventory of freely accessible nitrogen is atmospheric dinitrogen, but most organisms rely on more bioavailable forms of nitrogen, such as ammonium and nitrate, for growth The availability of these substrates depends on diverse nitrogen-transforming reactions that are carried out by complex networks of metabolically versatile microorganisms In this Review, we summarize our current understanding of the microbial nitrogen-cycling network, including novel processes, their underlying biochemical pathways, the involved microorganisms, their environmental importance and industrial applications

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The microbial nitrogen-cycling network

BACKGROUND The invention of the Haber-Bosch (H-B) process in the early 1900s to produce ammonia industrially from nitrogen and hydrogen revolutionized the manufacture of fertilizer and led to fundamental changes in the way food is produced. Its impact is underscored by the fact that about 50% of the nitrogen atoms in humans today originate from this single industrial process. In the century after the H-B process was invented, the chemistry of carbon moved to center stage, resulting in remarkable discoveries and a vast array of products including plastics and pharmaceuticals. In contrast, little has changed in industrial nitrogen chemistry. This scenario reflects both the inherent efficiency of the H-B process and the particular challenge of breaking the strong dinitrogen bond. Nonetheless, the reliance of the H-B process on fossil fuels and its associated high CO 2 emissions have spurred recent interest in finding more sustainable and environmentally benign alternatives. Nitrogen in its more oxidized forms is also industrially, biologically, and environmentally important, and synergies in new combinations of oxidative and reductive transformations across the nitrogen cycle could lead to improved efficiencies. ADVANCES Major effort has been devoted to developing alternative and environmentally friendly processes that would allow NH 3 production at distributed sources under more benign conditions, rather than through the large-scale centralized H-B process. Hydrocarbons (particularly methane) and water are the only two sources of hydrogen atoms that can sustain long-term, large-scale NH 3 production. The use of water as the hydrogen source for NH 3 production requires substantially more energy than using methane, but it is also more environmentally benign, does not contribute to the accumulation of greenhouse gases, and does not compete for valuable and limited hydrocarbon resources. Microbes living in all major ecosystems are able to reduce N 2 to NH 3 by using the enzyme nitrogenase. A deeper understanding of this enzyme could lead to more efficient catalysts for nitrogen reduction under ambient conditions. Model molecular catalysts have been designed that mimic some of the functions of the active site of nitrogenase. Some modest success has also been achieved in designing electrocatalysts for dinitrogen reduction. Electrochemistry avoids the expense and environmental damage of steam reforming of methane (which accounts for most of the cost of the H-B process), and it may provide a means for distributed production of ammonia. On the oxidative side, nitric acid is the principal commodity chemical containing oxidized nitrogen. Nearly all nitric acid is manufactured by oxidation of NH 3 through the Ostwald process, but a more direct reaction of N 2 with O 2 might be practically feasible through further development of nonthermal plasma technology. Heterogeneous NH 3 oxidation with O 2 is at the heart of the Ostwald process and is practiced in a variety of environmental protection applications as well. Precious metals remain the workhorse catalysts, and opportunities therefore exist to develop lower-cost materials with equivalent or better activity and selectivity. Nitrogen oxides are also environmentally hazardous pollutants generated by industrial and transportation activities, and extensive research has gone into developing and applying reduction catalysts. Three-way catalytic converters are operating on hundreds of millions of vehicles worldwide. However, increasingly stringent emissions regulations, coupled with the low exhaust temperatures of high-efficiency engines, present challenges for future combustion emissions control. Bacterial denitrification is the natural analog of this chemistry and another source of study and inspiration for catalyst design. OUTLOOK Demands for greater energy efficiency, smaller-scale and more flexible processes, and environmental protection provide growing impetus for expanding the scope of nitrogen chemistry. Nitrogenase, as well as nitrifying and denitrifying enzymes, will eventually be understood in sufficient detail that robust molecular catalytic mimics will emerge. Electrochemical and photochemical methods also demand more study. Other intriguing areas of research that have provided tantalizing results include chemical looping and plasma-driven processes. The grand challenge in the field of nitrogen chemistry is the development of catalysts and processes that provide simple, low-energy routes to the manipulation of the redox states of nitrogen.

http://science.sciencemag.org/content/sci/360/6391/eaar6611.full.pdf

Beyond fossil fuel-driven nitrogen transformations.

Glucose detection is of great significance in biomedical applications. Principles, methods and recent developments in electrochemical glucose sensors are reviewed here. Special attention is given to the discussion on some problems and bottlenecks in areas of nonenzymatic and enzymatic (glucose oxidase-based) amperometric glucose sensing.

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Recent advances in electrochemical glucose biosensors: a review

Superoxide, O2•–, is formed in all living organisms that come in contact with air, and, depending upon its biological context, it may act as a signaling agent, a toxic species, or a harmless intermediate that decomposes spontaneously Its levels are limited in vivo by two different types of enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD) Although superoxide has long been an important factor in evolution, it was not so when life first emerged on Earth at least 35 billion years ago At that time, the early biosphere was highly reducing and lacking in any significant concentrations of dioxygen (O2), very different from what it is today Consequently, there was little or no O2•– and therefore no reason for SOR or SOD enzymes to evolve Instead, the history of biological O2•– probably commences somewhere around 24 billion years ago, when the biosphere started to experience what has been termed the “Great Oxidation Event”, a transformation driven by the increase in O2 levels, formed by cyanobacteria as a product of oxygenic photosynthesis1 The rise of O2 on Earth caused a reshaping of existing metabolic pathways, and it triggered the development of new ones2 Its appearance led to the formation of the so-called “reactive oxygen species” (ROS), for example, superoxide, hydrogen peroxide, and hydroxyl radical, and to a need for antioxidant enzymes and other antioxidant systems to protect against the growing levels of oxidative damage to living systems


Dioxygen is a powerful four-electron oxidizing agent, and the product of this reduction is water




1


When O2 is reduced in four sequential one-electron steps, the intermediates formed are the three major ROS, that is, O2•–, H2O2, and HO•




2





3





4





5



Each of these intermediates is a potent oxidizing agent The consequences of their presence to early life must have been an enormous evolutionary challenge In the case of superoxide, we find the SOD and SOR enzymes to be widely distributed throughout current living organisms, both aerobic and anaerobic, suggesting that, from the start of the rise of O2 on Earth, the chemistry of superoxide has been an important factor during evolution


The SORs and three very different types of SOD enzymes are redox-active metalloenzymes that have evolved entirely independently from one another for the purpose of lowering superoxide concentrations SORs catalyze the one-electron reduction of O2•– to give H2O2, a reaction requiring two protons per superoxide reacted as well as an external reductant to provide the electron (eq 6) SODs catalyze the disproportionation of superoxide to give O2 and H2O2, a reaction requiring one proton per superoxide reacted, but no external reductant (eq 7)




6





7



All of the SOR enzymes contain only iron, while the three types of SODs are the nickel-containing SODs (NiSOD), the iron- or manganese-containing SODs (FeSOD and MnSOD), and the copper- and zinc-containing SODs (CuZnSOD) Although the structures and other properties of these four types of metalloenzymes are quite different, they all share several characteristics, including the ability to react rapidly and selectively with the small anionic substrate O2•– Consequently, there are some striking similarities between these otherwise dissimilar enzymes, many of which can be explained by considering the nature of the chemical reactivity of O2•– (see below)

Numerous valuable reviews describing the SOD and SOR enzymes have appeared over the years, but few have covered and compared all four classes of these enzymes, as we attempt to do here Thus, the purpose of this Review is to describe, compare, and contrast the properties of the SOR and the four SOD enzymes; to summarize what is known about their evolutionary pathways; and to analyze the properties of these enzymes in light of what is known of the inherent chemical reactivity of superoxide

Superoxide dismutases and superoxide reductases

Recent applications of carbon-based nanomaterials in analytical chemistry: critical review.

Ammonia (NH 3 )-oxidizing bacteria (AOB) emit substantial amounts of nitric oxide (NO) and nitrous oxide (N 2 O), both of which contribute to the harmful environmental side effects of large-scale agriculture. The currently accepted model for AOB metabolism involves NH 3 oxidation to nitrite (NO 2 – ) via a single obligate intermediate, hydroxylamine (NH 2 OH). Within this model, the multiheme enzyme hydroxylamine oxidoreductase (HAO) catalyzes the four-electron oxidation of NH 2 OH to NO 2 – . We provide evidence that HAO oxidizes NH 2 OH by only three electrons to NO under both anaerobic and aerobic conditions. NO 2 – observed in HAO activity assays is a nonenzymatic product resulting from the oxidation of NO by O 2 under aerobic conditions. Our present study implies that aerobic NH 3 oxidation by AOB occurs via two obligate intermediates, NH 2 OH and NO, necessitating a mediator of the third enzymatic step.

https://www.pnas.org/content/pnas/114/31/8217.full.pdf

Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase

Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N2O). It has been proposed that N2O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH2OH) quantitatively to N2O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH2OH to nitrite ( N O 2 − ) under aerobic conditions. Although we observe N O 2 − formation under aerobic conditions, its concentration is not stoichiometric with the NH2OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH2OH to N2O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an FeIII–NH2OH adduct of cyt P460 is oxidized to an {FeNO}6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH2OH, forming the N–N bond of N2O during a bimolecular, rate-determining step. We propose that N O 2 − results when nitric oxide (NO) dissociates from the {FeNO}6 intermediate and reacts with dioxygen. Thus, N O 2 − is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH2OH contributes to NO and N2O emissions from nitrifying microorganisms.

https://www.pnas.org/content/pnas/113/51/14704.full.pdf

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

Flavodiiron proteins (FDPs) catalyze reductive scavenging of dioxygen and nitric oxide in air-sensitive microorganisms. FDPs contain a distinctive non-heme diiron/flavin mononucleotide (FMN) active site. Alternative mechanisms for the nitric oxide reductase (NOR) activity consisting of either protonation of a diiron-bridging hyponitrite or "super-reduction" of a diferrous-dinitrosyl by the proximal FMNH(2) in the rate-determining step have been proposed. To test these alternative mechanisms, we examined a deflavinated FDP (deflavo-FDP) from Thermotoga maritima. The deflavo-FDP retains an intact diiron site but does not exhibit multiturnover NOR or O(2) reductase (O(2)R) activity. Reactions of the reduced (diferrous) deflavo-FDP with nitric oxide were examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. Anaerobic addition of nitric oxide up to one NO per diferrous deflavo-FDP results in formation of a diiron-mononitrosyl complex characterized by a broad S = (1)/(2 )EPR signal arising from antiferromagnetic coupling of an S = (3)/(2) {FeNO}(7) with an S = 2 Fe(II). Further addition of NO results in two reaction pathways, one of which produces N(2)O and the diferric site and the other of which produces a stable diiron-dinitrosyl complex. Both NO-treated and as-isolated deflavo-FDPs regain full NOR and O(2)R activities upon simple addition of FMN. The production of N(2)O upon addition of NO to the mononitrosyl deflavo-FDP supports the hyponitrite mechanism, but the concomitant formation of a stable diiron-dinitrosyl complex in the deflavo-FDP is consistent with a super-reduction pathway in the flavinated enzyme. We conclude that a diiron-mononitrosyl complex is an intermediate in the NOR catalytic cycle of FDPs.

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Insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme

Alternative Bioenergy: Updates to and Challenges in Nitrification Metalloenzymology

The unique active site of flavo-diiron proteins (FDPs) consists of a nonheme diiron-carboxylate site proximal to a flavin mononucleotide (FMN) cofactor. FDPs serve as the terminal components for reductive scavenging of dioxygen or nitric oxide to combat oxidative or nitrosative stress in bacteria, archaea, and some protozoan parasites. Nitric oxide is reduced to nitrous oxide by the four-electron reduced (FMNH2–FeIIFeII) active site. In order to clarify the nitric oxide reductase mechanism, we undertook a multispectroscopic presteady-state investigation, including the first Mossbauer spectroscopic characterization of diiron redox intermediates in FDPs. A new transient intermediate was detected and determined to be an antiferromagnetically coupled diferrous-dinitrosyl (S = 0, [{FeNO}7]2) species. This species has an exchange energy, J ≥ 40 cm–1 (JS1 ° S2), which is consistent with a hydroxo or oxo bridge between the two irons. The results show that the nitric oxide reductase reaction proceeds through succe...

http://pubs.acs.org/doi/pdf/10.1021/ja5022443

Jonathan D. Caranto

Papers

Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

Insights into the nitric oxide reductase mechanism of flavodiiron proteins from a flavin-free enzyme

Alternative Bioenergy: Updates to and Challenges in Nitrification Metalloenzymology

The Nitric Oxide Reductase Mechanism of a Flavo-Diiron Protein: Identification of Active-Site Intermediates and Products