IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ... IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Session Details: Monday 28 November 2011 Concurrent Session 2 – Function & Control of Nitrogenase 1530 - 1650 Authors: Lifen Yan 1 , Christie H. Dapper 2 , Simon J. George 1 , Hongxin Wang 1 , Devrani Mitra 1 , Weibing Dong 1 , Stephen P. Cramer 1 & William E. Newton 2 1 Department of Applied Science, University of California-Davis, Davis, CA 95616, USA. 2 Department of Biochemistry, Virginia Polytechnic Institute & State University, Blacksburg, VA 24061, USA Presentation Title: Carbon monoxide adducts of Azotobacter vinelandii Mo-nitrogenase Presentation Time: 1530 - 1550 Carbon monoxide (CO) is a non-competitive reversible inhibitor of all wild-type Mo-nitrogenase-catalyzed reactions except the reduction of protons to H2. An understanding of the CO interaction should give substantial insight into mechanism. Several CO-bound species are known; some reversibly interconvert. Which one is formed depends on the CO pressure (pCO) over Mo-nitrogenase during turnover. At pCO < 0.1 atm, “lo-CO” is formed, whereas under higher pCO, either “hi-CO” or “hi(5)-CO” or both are formed. Electron nuclear double resonance (ENDOR) spectroscopy suggests that: “lo-CO” has one bound CO bridging two Fe atoms; “hi-CO” has two CO ligands, each terminally bound to a different Fe atom; and “hi(5)-CO” has two bridging CO ligands. Both stopped-flow infrared and theoretical studies, however, appear inconsistent with these structures. To help resolve ambiguities, we investigated these species using photolysis under cryogenic conditions with Fourier transform-infrared (FT-IR) detection. Photolysis of “hi-CO” indicates loss of terminally bound (1973 cm -1 ) and bridging (1679 cm -1 ) CO molecules, with concomitant formation of a species with one bridging (1711 cm -1 ) CO molecule. These assignments were confirmed by using isotopically labeled CO. Our results are therefore only partly consistent with the ENDOR assignments. We extended these studies to two altered Mo-nitrogenases, both with a substitution at α-histidine-195 in the MoFe protein, namely α-H195Q and α-H195N. Surprisingly, although we expected a similar band at ca. 1973 cm -1 on photolysis of α-H195N, we saw a band at lower energy (1936 cm -1 ). More surprisingly, photolysis of α-H195Q showed both CO-related bands, at 1969 and 1932 cm -1 . Structures will be proposed for these species and the results will be discussed in terms of both CO inhibition and the catalytic mechanism. 26 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Session Details: Monday 28 November 2011 Concurrent Session 2 – Function & Control of Nitrogenase 1530 - 1650 Authors: He Wang, Pedro Filipe Teixeira, Tiago T. Selão, Agneta Norén, Catrine L. Berthold, Martin Högbom, Stefan Nordlund Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Sweden. Presentation Title: Regulation of Nitrogenase activity in Rhodospirillum rubrum – an interplay of PII proteins, DRAT, DRAG and membrane sequestration Presentation Time: 1550 - 1610 In the Rhodospirillum rubrum and some other phototrophic bacteria, nitrogenase activity is regulated at the metabolic level in response to changes in nitrogen availability or in energy supply, i.e. light/darkness. This regulation is also present in some species of Azospirillum, although in that case the energy supply is reflected as the concentration of oxygen. At the molecular level this regulation is due to reversible ADP-ribosylation of the Feprotein. DRAT catalyzes the addition of an ADP-ribose from NAD + and DRAG catalyzes the removal of ADPribose moiety, thereby restoring activity. One of the major questions has been the identity of the signal(s) in this system and some years ago we proposed that the association of DRAG to the chromatophore membrane plays a central role in the regulatory mechanism. Since then we and a number of other groups have provided evidence supporting this model and also demonstrated the involvement of AmtB1 and PII proteins in this regulation. There are three PII paralogs in R. rubrum, GlnB, GlnJ and GlnK. GlnB and GlnJ have both been shown to have specific functions in the regulation of nitrogen metabolism, whereas no specific function has yet been identifíed for GlnK. We have now furthered our studies on the role of the interaction of DRAG with a partner in the chromatophore membrane in the regulation of nitrogenase activity and the involvement of PII proteins in this interaction as well as the regulation of DRAT. We have also demonstrated the influence of different nitrogen sources during growth, N2 or glutamate, on the pathway leading to regulation of nitrogenase activity, both in ammonium and energy “switch-off”. Based on our studies we propose a model explaining the communication within the cell leading to a concerted regulation of nitrogen assimilation and nitrogen fixation. 27 2011
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17 th <strong>International</strong> Congress on <strong>Nitrogen</strong> <strong>Fixation</strong><br />
Fremantle, Western Australia<br />
27 November – 1 December 2011<br />
Session Details: Monday 28 November 2011<br />
Concurrent Session 2 – Function & Control of <strong>Nitrogen</strong>ase<br />
1530 - 1650<br />
Authors: He Wang, Pedro Filipe Teixeira, Tiago T. Selão, Agneta Norén, Catrine L. Berthold, Martin<br />
Högbom, Stefan Nordlund<br />
Department of Biochemistry and Biophysics, Stockholm University, SE-10691, Sweden.<br />
Presentation Title: Regulation of <strong>Nitrogen</strong>ase activity in Rhodospirillum rubrum – an interplay of PII proteins,<br />
DRAT, DRAG and membrane sequestration<br />
Presentation Time: 1550 - 1610<br />
In the Rhodospirillum rubrum and some other phototrophic bacteria, nitrogenase activity is regulated at the<br />
metabolic level in response to changes in nitrogen availability or in energy supply, i.e. light/darkness. This<br />
regulation is also present in some species of Azospirillum, although in that case the energy supply is reflected as<br />
the concentration of oxygen. At the molecular level this regulation is due to reversible ADP-ribosylation of the Feprotein.<br />
DRAT catalyzes the addition of an ADP-ribose from NAD + and DRAG catalyzes the removal of ADPribose<br />
moiety, thereby restoring activity. One of the major questions has been the identity of the signal(s) in this<br />
system and some years ago we proposed that the association of DRAG to the chromatophore membrane plays a<br />
central role in the regulatory mechanism. Since then we and a number of other groups have provided evidence<br />
supporting this model and also demonstrated the involvement of AmtB1 and PII proteins in this regulation.<br />
There are three PII paralogs in R. rubrum, GlnB, GlnJ and GlnK. GlnB and GlnJ have both been shown to have<br />
specific functions in the regulation of nitrogen metabolism, whereas no specific function has yet been identifíed<br />
for GlnK.<br />
We have now furthered our studies on the role of the interaction of DRAG with a partner in the chromatophore<br />
membrane in the regulation of nitrogenase activity and the involvement of PII proteins in this interaction as well<br />
as the regulation of DRAT. We have also demonstrated the influence of different nitrogen sources during growth,<br />
N2 or glutamate, on the pathway leading to regulation of nitrogenase activity, both in ammonium and energy<br />
“switch-off”. Based on our studies we propose a model explaining the communication within the cell leading to a<br />
concerted regulation of nitrogen assimilation and nitrogen fixation.<br />
27<br />
2011
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