IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...
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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: Lifen Yan 1 , Christie H. Dapper 2 , Simon J. George 1 , Hongxin Wang 1 , Devrani Mitra 1 ,<br />
Weibing Dong 1 , Stephen P. Cramer 1 & William E. Newton 2<br />
1 Department of Applied Science, University of California-Davis, Davis, CA 95616, USA.<br />
2 Department of Biochemistry, Virginia Polytechnic Institute & State University, Blacksburg,<br />
VA 24061, USA<br />
Presentation Title: Carbon monoxide adducts of Azotobacter vinelandii Mo-nitrogenase<br />
Presentation Time: 1530 - 1550<br />
Carbon monoxide (CO) is a non-competitive reversible inhibitor of all wild-type Mo-nitrogenase-catalyzed<br />
reactions except the reduction of protons to H2. An understanding of the CO interaction should give substantial<br />
insight into mechanism. Several CO-bound species are known; some reversibly interconvert. Which one is<br />
formed depends on the CO pressure (pCO) over Mo-nitrogenase during turnover. At pCO < 0.1 atm, “lo-CO” is<br />
formed, whereas under higher pCO, either “hi-CO” or “hi(5)-CO” or both are formed. Electron nuclear double<br />
resonance (ENDOR) spectroscopy suggests that: “lo-CO” has one bound CO bridging two Fe atoms; “hi-CO” has<br />
two CO ligands, each terminally bound to a different Fe atom; and “hi(5)-CO” has two bridging CO ligands. Both<br />
stopped-flow infrared and theoretical studies, however, appear inconsistent with these structures. To help<br />
resolve ambiguities, we investigated these species using photolysis under cryogenic conditions with Fourier<br />
transform-infrared (FT-IR) detection. Photolysis of “hi-CO” indicates loss of terminally bound (1973 cm -1 ) and<br />
bridging (1679 cm -1 ) CO molecules, with concomitant formation of a species with one bridging (1711 cm -1 ) CO<br />
molecule. These assignments were confirmed by using isotopically labeled CO. Our results are therefore only<br />
partly consistent with the ENDOR assignments. We extended these studies to two altered Mo-nitrogenases,<br />
both with a substitution at α-histidine-195 in the MoFe protein, namely α-H195Q and α-H195N. Surprisingly,<br />
although we expected a similar band at ca. 1973 cm -1 on photolysis of α-H195N, we saw a band at lower energy<br />
(1936 cm -1 ). More surprisingly, photolysis of α-H195Q showed both CO-related bands, at 1969 and 1932 cm -1 .<br />
Structures will be proposed for these species and the results will be discussed in terms of both CO inhibition and<br />
the catalytic mechanism.<br />
26<br />
2011