The need for an alternative to red cells in transfusions has led to the creation of hemoglobin-based oxygen carriers (HBOCs). However, evaluations of all products tested in clinical trials have noted cardiovascular complications, raising questions about their safety that led to the abandonment of all those products. It has been considered that the adverse side effects come from the scavenging of the vasodilator – nitric oxide (NO) by the deoxyheme sites of the hemoglobin derivatives. Another observation is that HBOCs with lower oxygen affinity than red cells release oxygen prematurely in arterioles, triggering an unwanted homeostatic response. Since the need for such a product remains critical, it is important to understand the reactivity patterns that contribute to the observed complications. Various alterations of the protein have been attempted in order to reduce HBOC-induced vasoconstriction. Recent reports suggest that a safe and effective product should be pure, homogenous and have a high molecular weight along with appropriate oxygenation properties. While these properties are clearly important, vasodilatory features of hemoglobin through its nitrite reductase activity may also act as an in situ source of NO. It follows that HBOCs with an enhanced ability to produce NO from endogenous nitrite may serve to counteract vasoactivity associated with NO-scavenging by hemoglobin. Here we characterize the effects of different protein modifications on the nitrite reductase activity of hemoglobin. We produced a variety of HBOCs that include cross-linked tetramers, polyethylene glycol (PEG) conjugates and bis-tetramers of hemoglobin. We report that the rate of NO production strongly depends on the conformational state of the protein, with R-state stabilized proteins (PEG-Hbs), exhibiting the fastest rates. In particular, we found that PEGylated bis-tetramers of hemoglobin (BT-PEG) exhibit increased nitrite reductase activity while retaining cooperativity and stability. Animal studies of BT-PEG demonstrated that this material is benign: it did not cause significant increases in systemic blood pressure in mice, the major side effect associated with existing HBOCs. BT-PEG exhibits an enhanced nitrite reductase activity together with sample purity and homogeneity, molecular size and shape, and appropriate oxygenation properties, characteristics of a clinically useful HBOC.

No description available.

<p>Cytochrome <i>cd</i>₁ nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the <i>d</i>₁ haem iron of the enzyme.</p><p>Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions.</p><p>Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction.</p><p>Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty.</p><p>Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.</p>

Cytochrome cd1 nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the d1 haem iron of the enzyme. Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions. Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction. Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty. Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.

No description available.

El óxido nítrico (NO) es una molécula gaseosa altamente reactiva que regula el crecimiento y el desarrollo de las plantas así como sus respuestas de defensa. El NO se produce principalmente a partir de nitrito por las nitrato reductasas (NRs) en balance con las nitrito reductasas (NiRs), y es percibido a través de un mecanismo en el que está involucrada la proteólisis dirigida por la secuencia aminoterminal del grupo VII de los factores de transcripción ERF (ERFVIIs). El NO ejerce especialmente su función señalizadora al causar modificaciones postraduccionales en las proteínas y alterar su función, estructura y/o estabilidad. Por estos medios y en colaboración con distintas rutas de señalización fitohormonales, el NO es capaz de regular un amplio abanico de procesos celulares en plantas, incluyendo aquellos relacionados con la adquisición de tolerancia a la congelación. Utilizando Arabidopsis thaliana como planta modelo, en este trabajo se descubrió que el NO puede regular su propia biosíntesis, puesto que las enzimas NRs y NiRs fueron reguladas por tres factores principales: señalización inducida por nitrato y controlada por la función del factor de transcripción NIN-like protein 7 (NLP7), la proteólisis dirigida por la secuencia aminoterminal, y la degradación mediada por el proteasoma, probablemente ocasionada por modificaciones postraduccionales relacionadas con el NO. Adicionalmente, se descubrió que el factor de transcripción ERFVII RAP2.3 regula negativamente tanto la biosíntesis de NO como las respuestas que desencadena a través de un mecanismo similar a un reóstato en el que están involucradas ramas específicas relacionadas con el NO de las rutas de señalización de jasmonato y ácido abscísico. Por otro lado, una caracterización metabolómica y transcriptómica combinada de plantas mutantes nia1,2noa1-2 deficientes en NO y plantas fumigadas con NO permitió desentrañar una serie de mecanismos que están controlados por NO. En primer lugar, la percepción de NO en los hipocotilos requeriría varias hormonas para ser completada, como fue confirmado por los rastreos de acortamiento de hipocotilo por NO con mutantes relacionados con hormonas y la colección TRANSPLANTA de líneas transgénicas que expresan condicionalmente factores de transcripción de Arabidopsis. En segundo lugar, dosis elevadas de NO causan una reprogramación masiva aunque transitoria de los metabolismos primario y secundario, incluyendo la alteración del estado redox celular, la alteración de la permeabilidad de estructuras lipídicas y el recambio de proteínas y ácidos nucleicos. Por último, se descubrió que el NO previene el desarrollo de la tolerancia a congelación bajo condiciones no estresantes de temperatura, mientras que resulta esencial para la aclimatación a frío desencadenada por bajas temperaturas que conduce a una tolerancia mejorada a congelación. El NO conseguiría esta modulación afinada de la activación de respuestas relacionadas con frío al coordinar la acumulación de diferentes metabolitos y hormonas. En conjunto, este trabajo arroja luz sobre los mecanismos mediante los cuales, al interactuar con varias rutas señalizadoras y metabólicas, el NO puede regular distintos procesos clave de la fisiología vegetal. / L'òxid nítric (NO) és una molècula gasosa altament reactiva que regula el creixement i desenvolupament de les plantes així com les seves respostes de defensa. El NO es produeix principalment a partir de nitrit per les nitrat reductases (NRs) en balanç amb les nitrit reductases (NiRs), i és percebut a traves d'un mecanisme que inclou la proteòlisi dirigida per la seqüència aminoterminal del grup VII dels factors de transcripció ERF (ERFVII). El NO exerceix la seva funció senyalitzadora majoritàriament al provocar modificacions postraduccionals en les proteïnes i alterar la seva funció, estructura i/o estabilitat. Mitjançant aquestes modificacions i en col·laboració amb distintes rutes de senyalització fitohormonals, el NO es capaç de regular un ampli espectre de processos cel·lulars en plantes, inclosos aquells relacionats amb l'adquisició de tolerància a la congelació. Emprant Arabidopsis thaliana com a planta model, en aquest treball es va descobrir que el NO regula la seva pròpia biosíntesi, donat que els enzims NRs i NiRs foren regulades per tres factors principals: senyalització induïda per nitrat i controlada per la funció del factor de transcripció NIN-like protein 7 (NLP7), la proteòlisi dirigida per la seqüència aminoterminal, i la degradació mitjançant el proteasoma, probablement a causa de modificacions postraduccionals relacionades amb el NO. A més, es va descobrir que el factor de transcripció ERFVII RAP2.3 regula negativament tant la biosíntesi de NO com les respostes que desencadena aquest a través d'un mecanisme similar a un reòstat en el que estan involucrades branques específiques de les rutes de senyalització de jasmonat i àcid abscísic relacionades amb el NO. Per altre costat, una caracterització metabolòmica i transcriptòmica combinada de plantes mutants nia1,2noa1-2 deficients en NO i plantes fumigades amb NO va permetre desentranyar una sèrie de mecanismes que estan controlats per NO. En primer lloc, la percepció de NO en els hipocòtils requeriria de varies hormones, com fou confirmat pels rastrejos d'acurtament d'hipocòtil per NO amb mutants relacionats amb hormones i la col·lecció TRANSPLANTA de línies transgèniques d'expressió condicional de factors de transcripció d'Arabidopsis. En segon lloc, dosis elevades de NO causen una reprogramació massiva, encara que transitòria, dels metabolismes primari i secundari, incloent l'alteració de l'estat redox cel·lular, canvis en la permeabilitat de estructures lipídiques i el recanvi de proteïnes i àcids nucleics. Per últim, es va descobrir que el NO prevé el desenvolupament de la tolerància a congelació en condicions no estressants de temperatura, mentre que resulta essencial per a l'aclimatació a fred induïda per baixes temperatures que condueix a una tolerància millorada a congelació. El NO aconseguiria aquesta modulació minuciosa de l'activació de les respostes relacionades amb fred al coordinar l'acumulació de diferents metabòlits i hormones. En conjunt, aquest treball clarifica els mecanismes pels quals el NO pot regular distints processos clau de la fisiologia vegetal al interactuar amb varies rutes senyalitzadores i metabòliques. / Nitric oxide (NO) is a highly reactive gaseous molecule that regulates plant growth and development as well as defense responses. NO is mainly produced from nitrite by nitrate reductases (NRs) in balance with nitrite reductases (NiRs), and is sensed through a mechanism involving the N-end rule pathway-mediated proteolysis of the group VII of ERF transcription factors (ERFVIIs). NO especially exerts its signaling function by triggering post-translational modifications in proteins and altering their function, structure and/or stability. By these means and in collaboration with different phytohormone signaling pathways, NO is capable of regulating a wide array of cell processes in plants, including those related to the acquirement of freezing tolerance. By using Arabidopsis thaliana as model plant, during the development of this work it was found that NO can regulate its own biosynthesis, as NRs and NiR enzymes were regulated by three main factors: nitrate-induced signaling controlled by the function of the NIN-like protein 7 (NLP7) transcription factor, N-end rule proteolytic pathway, and proteasome-mediated degradation, likely triggered by NO-related post-translational modifications. In addition, the ERFVII transcription factor RAP2.3 was found to negatively regulate both the NO biosynthesis and their triggered responses through a rheostat-like mechanism that involves specific NO-related branches of jasmonate and abscisic acid signaling pathways. On the other hand, a combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1-2 mutant plants and NO-fumigated plants allowed to unravel a number of mechanisms that are controlled by NO. First, NO perception in hypocotyls would require various hormones to be fulfilled as it was confirmed by NO-triggered hypocotyl shortening screenings with hormone-related mutants and the TRANSPLANTA collection of transgenic lines conditionally expressing Arabidopsis transcription factors. Second, high NO doses caused a massive but transient reprogramming of primary and secondary metabolism, including alteration of the cellular redox status, alteration of the permeability of lipidic structures or turnover of proteins and nucleic acids. Lastly, NO was found to prevent the development of freezing tolerance under non-stress temperature conditions, while being essential for the low temperature stress-triggered cold acclimation that leads to enhanced freezing tolerance. NO would achieve this fine-tuned modulation of the activation of the cold-related responses by coordinating the accumulation of different metabolites and hormones. Altogether, this work sheds light on the mechanisms by which, by interacting with various signaling and metabolic pathways, NO can regulate several key processes of plant physiology. / Costa Broseta, Á. (2018). Regulation of the nitric oxide synthesis and signaling by posttranslational modifications and N-end rule pathway-mediated proteolysis in Arabidopsis thaliana [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/114825

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.