Orientador: Ladaslav Sodek / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T05:22:48Z (GMT). No. of bitstreams: 1 Souza_SarahCarolineRibeiro_D.pdf: 2548021 bytes, checksum: 61437f1032c9e4fcc9ee2131a8f6dabe (MD5) Previous issue date: 2014 / Resumo: A soja é uma leguminosa amplamente utilizada para estudos envolvendo a fixação biológica de Nitrogênio (N), seja por sua grande importância econômica, seja por sua elaborada e bem sucedida relação simbiótica com rizóbios do gênero Bradyrhizobium, sendo capaz de obter todo N necessário para seu desenvolvimento através da fixação do N2 atmosférico. Todavia, o metabolismo de N em plantas de soja noduladas é sensível à hipóxia provocada pelo alagamento do sistema radicular. Dessa forma, este trabalho teve por objetivo, avaliar os efeitos do alagamento sobre o metabolismo de N em nódulos de soja em diferentes períodos de inundação e recuperação após a drenagem. Para isto, plantas de soja noduladas com o B. elkanii foram submetidas aos experimentos de inundação e recuperação, sendo os períodos de inundação/recuperação variáveis de acordo com o experimento. As alterações no metabolismo foram avaliadas através da análise da composição de aminoácidos por HPLC e avaliação da incorporação de 15N2 nos aminoácidos dos nódulos. Também foi avaliada a atividade da nitrogenase e a expressão dos genes nifH e nifD (nitrogenase), e dos genes que codificam as enzimas glutamato descarboxilase (GAD) e asparagina sintetase (AS) em soja. Verificamos que asparagina (ASN) é o aminoácido mais abundante em nódulos de soja (50%), seguido por glutamato (GLU), serina (SER) e ácido gama-aminobutírico (GABA). Com a inundação observou-se principalmente, uma redução acentuada de ASN, e aumento de GABA, após 4 dias (quando ASN reduziu a quase 1%), além de pequenas alterações na composição de outros aminoácidos. Com os tratamentos de recuperação ASN recuperou-se lentamente e quanto maior o período de exposição ao estresse mais lento o período de recuperação. Aparentemente a redução de ASN nos nódulos foi compensada pelo aumento de GABA. A atividade da nitrogenase foi fortemente inibida pela inundação, mas se recuperou totalmente. Quanto à incorporação de 15N2, verificamos que GLN foi o aminoácido marcado em grau mais elevado, seguido respectivamente por GLU, ASP, ALA e SER. A marcação dos aminoácidos ASN e GABA foi baixa, e isso pode ser devido ao grande "pool" destes aminoácidos no nódulo, ou pela entrada destes aminoácidos a partir de uma fonte não-marcada como o floema. Com relação à inundação, observou-se uma redução na incorporação de 15N2 em ASN, e a recuperação também foi lenta, também houve redução na incorporação em outros aminoácidos como ASP e GLN. A hipóxia afetou a expressão dos genes avaliados nos nódulos. Houve uma redução na expressão dos genes AS1 e AS2, o que condiz com a redução nos teores de ASN. O gene que codifica a GAD também foi menos expresso em nódulos submetidos à inundação o que não explica o aumento de GABA no nódulo durante o estresse. A expressão dos genes nifH e nifD também diminuiram com a inundação, mas se recuperam, e condizem com o observado para atividade da nitrogenase. Dessa forma, verificamos que a inundação afeta o metabolismo de N nos nódulos de soja, em diversos aspectos, como a composição de aminoácidos, atividade da nitrogenase e na expressão de genes envolvidos na assimilação do N em aminoácidos nos nódulos / Abstract: Soybean has been widely used in studies of biological nitrogen fixation, not only because of its economic importance, but in view of its highly efficient symbiotic relationship with rhizobia of the genus Bradyrhizobium, which can supply all the N needed for full development of the plant. However, the process is highly sensitive to oxygen deficiency provoked by waterlogging of the root system, resulting in a rapid and strong inhibition of nitrogen fixation since the availability of oxygen for nitrogenase activity is tightly controlled in the nodule and close to limiting under normal conditions. Thus, this study aimed to evaluate the effects of flooding on the N metabolism in nodules of soybean in different periods of flooding and recovery after drainage. For this, soybean plants nodulated with B. elkanii were subjected to flooding and recovery experiments at stages V7/V8 The flooding/recovery duration was where the flooding/recovery periods were variable according to the experiment. Changes in metabolism were evaluated by analyzing the amino acid composition by HPLC and by assessing the amino acid incorporation of 15N2 of the nodules. Nitrogenase activity and expression of nifH and nifD (nitrogenase) genes, and genes encoding GAD and AS in soybean were also evaluated. The most abundant amino acid in soybean nodules was asparagine (ASN) (50%), followed by glutamate (GLU), serine (SER) and gama-aminobutyric acid (GABA). On flooding, there was a marked decrease of ASN, and increased GABA, mainly after 4 days when ASN dropped to near 1%, as well as smaller alterations in the composition of other amino acids. With the recovery treatments, ASN recovered slowly and the longer the period of exposure to the stress the longer the recovery period. It appears that the reduction of the ASN in nodules is compensated by the increase of GABA. The nitrogenase activity was strongly inhibited by flooding, but full recovery was possible. Regarding the incorporation of 15N2, it was found that GLN is the amino acid labelled to the highest degree, followed respectively by GLU, ASP, ALA and SER. The labelling of the amino acids GABA and ASN was low, which may be due either to the large pool of these amino acids in the nodule, or to the entry of these amino acids from a non-labelled source such as the phloem. Flooding resulted in a reduction of the incorporation of 15N2 in ASN, and recovery was also slow. There was also reduction in the incorporation of 15N2 in other amino acids, such as Asp and GLN. Hypoxia affected the expression of all genes evaluated in nodules. There was a reduction in the expression of the AS1 and AS2 genes, which is consistent with the fall in levels of ASN. Recovery of expression was slow and gradual. Expression of the gene encoding the enzyme GAD was also strongly suppressed in nodules under flooding which does not therefore explain the increase of GABA in the nodule during stress. The expression of nifH and nifD genes were also strongly decreased on flooding, but recovered fully, consistent with the observed data for nitrogenase activity. In conclusion, it was found that flooding affects the metabolism of N in soybean nodules, in diverse ways, such as the amino acid composition, nitrogenase activity, and the expression of genes involved in N assimilation of nodule amino acids / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal

Nitrogenase is a metalloenzyme that plays a key role in biological nitrogen fixation by catalysing the reduction of dinitrogen to ammonia. Study of nitrogenase is particularly challenging because of its unique electron transfer and catalytic components. This Thesis describes the development of a mediated electron transfer system for the MoFe protein of nitrogenase, in order to overcome the complexity of electron transfer by the native reductant Fe protein coupled to hydrolysis of ATP. A series of redox mediators was employed including Eu^III/II-polyaminocarboxylate complexes, which have reduction potentials in a very negative range. In the presence of the redox mediators, the wild type MoFe protein exhibits a catalytic current due to protein-catalysed proton reduction. With this mediated electron transfer method, the potential of proton reduction by nitrogenase was determined for the first time. The redox mediator system was also applied in an infrared (IR) spectroelectrochemical study of CO binding to the wild type and β-98^His variant MoFe protein. The first IR evidence was provided for ATP-independent CO binding to the active site of the MoFe protein, in both the wild type and the variant. The peak wavenumbers and time-dependent changes in intensity found in this study are consistent with the result of previous CO coordination with nitrogenase obtained by electron transfer from the Fe protein driven by ATP. This strongly suggests that this mediated electron transfer approach can deliver low potential electrons into the MoFe protein and reduce the active site FeMoco to the substrate binding level. Moreover, this technique allows electrocatalytic activity of the protein to be monitored and the change in redox activity can be correlated directly to the potential. With the same technique, a study of cyanide binding was performed on different variant MoFe proteins of nitrogenase. The redox properties of the isolated cofactor of Mo- and V-dependent nitrogenase were investigated in parallel to the study of the protein-bound cofactors. It was found that FeVco, the active site from V-nitrogenase, exhibited different redox properties compared to that of Mo-nitrogenase. This might account for the unexpected activity in CO reduction that was reported previously for V-nitrogenase.

Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown. By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, could account for the differences in catalytic properties depicted by these enzymes. The quaternary structure of the dinitrogenases also differs. Such component in the Mo-nitrogenase is an α2β2 tetramer while for the V- an Fe-only nitrogenase is an α2β2δ2 hexamer. The latter enzymes are unable to reduce N2 in the absence of a functional δ subunit, yet they reduce H+ and the non-physiological substrate C2H2. Therefore, the δ subunit is essential for V- and Fe-only dependent nitrogen fixation by a mechanism that still remains unknown. In attempt to understand why the δ subunit is essential for V-dependent N2 reduction from a structural stand point, this work presents the strategy followed to clone the vnfG gene and purify its expression product, the δ subunit. The purified protein was subjected to crystallization trials and used to stabilize a histidine-tagged VFe protein that would otherwise purify with low Fe2+ content and poor H+ and C2H2 reduction activities. The VFe preparation was used to conduct substrate reduction assays to assess: i) The electron allocation patterns to each of the reduction products of the substrates C2H2, N2, N2H4, and N3−; and ii) Inhibition patterns among substrate and inhibitor of the nitrogenase reaction. This work also reports on the effect N2H4 and N3− has on the electron flux to the products of the C2H2 reduction. The work presented herein provides information with which to compare and contrast biological nitrogen fixation as catalyzed by the Mo- and V-nitrogenases from Azotobacter vinelandii.

Nitrogenase is the enzyme that catalyzes the reduction of nitrogen to ammonia in a reaction requiring MgATP hydrolysis. Two component proteins of nitrogenase are the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein). Nitrogenase contains two nucleotide binding sites. During catalysis, the Fe protein binds two MgATP first. The confonnational changes induced upon MgA TP binding allow the Fe protein to associate with the MoFe protein. After the formation of the Fe protein-MoFe protein complex, a single electron is transferred from the Fe protein to the MoFe protein, an event that is coupled to MgATP hydrolysis in the Fe protein. The wild-type Fe protein and all the altered Fe proteins studied so far are homodimeric. In order to assess the contribution of each nucleotide binding site in the Fe protein to the events occurring during nitrogenase catalysis, a heterodimeric Fe protein was constructed that has Asp 39 substituted by Asn in one subunit and the other subunit the same as the wild-type Fe protein. Characterization of this heterodimeric Fe protein showed that alterations in the properties of the [4Fe-4S] cluster that occur upon nucleotide binding to the Fe protein are due to the additive effect of each nucleotide binding to the Fe protein. The rates of MgATP hydrolysis and MgATP-dependent primary electron transfer of this heterodimeric Fe protein are intermediate between those of the homodimeric wild-type Fe protein and D39N Fe protein. These observations suggested that each ATP binding site contributes to the rate acceleration of primary electron transfer. After electron transfer, this heterodimeric Fe protein forms a tight complex with the MoFe protein, demonstrating that alteration in one subunit is enough for the formation of a tight nitrogenase complex. When this heterodimeric Fe protein was combined with the MoFe protein, no substrate reduction was detected. Therefore, two functional subunits of the Fe protein are necessary for reduction of substrates. The mechanism of ATP hydrolysis in the Fe protein was also investigated. Using site-directed mutagenesis, the role of lysine 10 of the Azotobacter vinelandii nitrogenase Fe protein in MgATP hydrolysis was examined. Changing Lys 10 of the protein to Arg resulted in an Fe protein that hydrolyzed MgATP at a rate 3% that of the wild-type Fe protein. The affinities of the K10R Fe protein for nucleotides and the changes in the properties of the [4Fe-4S] cluster of the K10R Fe protein upon nucleotide binding were compared with those of the wild-type Fe protein. These results indicated that in the absence of the MoFe protein, the interactions of the Kl OR Fe protein with nucleotides are similar to the wild-type Fe protein. After the Fe protein-MoFe protein complex formation, the dramatic decrease in the rate of MgATP hydrolysis of the K10R Fe protein indicated a role of Lys 10 in ATP hydrolysis. This conclusion is consistent with the X-ray crystal structure of the nitrogenase complex stabilized by the AlF4-•ADP, where Lys 10 is proposed to facilitate product formation in ATP hydrolysis.

Thesis (Ph.D.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23956号 / 農博第2505号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5391(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 栗原 達夫, 教授 小川 順, 准教授 黒田 浩一 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [90]-114).

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: John W. Peters. Includes bibliographical references (leaves 131-147).

The reduction of dinitrogen (N2) to ammonia (NH3) will continue to play a vital role in society as the population of the world grows and maintains its dependence on artificial fertilizers. This energy-intensive transformation is achieved industrially by the Haber-Bosch process and naturally via nitrogenase enzymes. Recent synthetic systems attempt to produce NH3 artificially but with lower energy costs than Haber-Bosch by modeling their designs after nitrogenase. This dissertation describes the progress made in one iron-phosphine system, the water-soluble Fe(DMeOPrPE)2N2, capable of producing NH3 at room temperature and pressure. Chapter I describes the history of the coordination chemistry of N2 to a variety of metals, with a focus on iron complexes. In addition to exploring the range of coordination geometries and supporting ligands of such complexes, the application of N2 coordination complexes towards NH3 formation is analyzed. Chapter II discusses the various methods for quantifying yields of ammonia. Along with a historical perspective on the popular indophenol method, the challenges and best conditions for measuring NH3 in the Fe-DMeOPrPE system are defined. Chapter III explores a series of trans-hydrido intermediates along a potential protonation pathway of Fe(DMeOPrPE)2N2. The complete series of reduced dinitrogen ligands (N2, N2H2, N2H4, and NH3) on the Fe(DMeOPrPE)2H+ scaffold is described. Chapter IV highlights the discovery and characterization of a unique bridged Fe(I) dimer, observed during the protonation of Fe(DMeOPrPE)2N2 as a dark purple intermediate. Chapter V describes the electrochemistry of certain intermediates in the Fe-DMeOPrPE system. This insight should open new avenues for future investigations. By altering the electronics of the system, more NH3 may eventually be produced. Chapter VI provides a summary of this work. This dissertation includes previously published and unpublished co-authored material. / 10000-01-01

Nitrogen is a critical nutrient for growth and reproduction in living organisms. Although the Earth’s atmosphere is composed of ~80% nitrogen gas (N2), it is inaccessible to most living organisms in that form. Biological nitrogen fixation, however, can be performed by microbes that harbor the enzyme nitrogenase. This enzyme converts N2 into bioavailable ammonia (NH3) and accounts for at least half of the “fixed”nitrogen on the planet. The other major contributor to ammonia production is the industrial Haber-Bosch process. While the Haber-Bosch process has made significant advances in sustaining the global food supply through the generation of fertilizer, it requires high temperature and pressure and fossil fuels. This makes nitrogenase an ideal system for study, as it is capable of performing this challenging chemistry under ambient conditions and without fossil fuels. Nitrogenase requires energy and electrons to convert N2into NH3. The work presented here examined how the enzyme receives electrons to perform the reaction. It was discovered that some microbes employ a novel mechanism that adjusts the energy state of the electrons so that nitrogenase can accept them. Further, the slowest step that takes place in nitrogenase once the electrons are taken up was identified. Finally, by capitalizing on fundamental knowledge, a biohybrid system was designed to grow nitrogen-fixing bacteria in association with electrodes for light-driven production of fixed nitrogen that has potential to be used as a fertilizer for plant growth. Gaining an in-depth understanding of nitrogenase provides insight into one of the most challenging biological reactions, and the newfound knowledge may be a catalyst in developing more efficient systems for sustainable ammonia production.