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IR-Monitored Photolysis of CO-Inhibited Nitrogenase: A Major EPR-Silent Species with Coupled Terminal CO Ligands

Authors

  • Lifen Yan,

    1. Department of Chemistry, University of California, Davis, CA 95616 (USA)
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  • Dr. Vladimir Pelmenschikov,

    Corresponding author
    1. Institut für Chemie, Technische Universität Berlin, 10623 Berlin (Germany)
    • Vladimir Pelmenschikov, Institut für Chemie, Technische Universität Berlin, 10623 Berlin (Germany)===

      Stephen P. Cramer, Department of Chemistry, University of California, Davis, CA 95616 (USA)===

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  • Christie H. Dapper,

    1. Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (USA)
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  • Aubrey D. Scott,

    1. Department of Chemistry, University of California, Davis, CA 95616 (USA)
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  • Prof. William E. Newton,

    1. Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 (USA)
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  • Prof. Stephen P. Cramer

    Corresponding author
    1. Department of Chemistry, University of California, Davis, CA 95616 (USA)
    2. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (USA)
    • Vladimir Pelmenschikov, Institut für Chemie, Technische Universität Berlin, 10623 Berlin (Germany)===

      Stephen P. Cramer, Department of Chemistry, University of California, Davis, CA 95616 (USA)===

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Abstract

Fourier transform infrared spectroscopy (FTIR) was used to observe the photolysis and recombination of a new EPR-silent CO-inhibited form of α-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K reveals a strong negative IR difference band at equation image=1938 cm−1, along with a weaker negative feature at 1911 cm−1. These bands and the associated chemical species have both been assigned the label “Hi-3”. A positive band at equation image=1921 cm−1 was assigned to the “Lo-3” photoproduct. By using an isotopic mixture of 12C 16O and 13C 18O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at approximately equation image=1917 cm−1. Although in previous studies Lo-3 was not observed to recombine, by extending the observation range to 200–240 K, we found that recombination to Hi-3 does indeed occur, with an activation energy of approximately 6.5 kJ mol−1. The frequencies of the Hi-3 bands suggest terminal CO ligation. This hypothesis was tested with DFT calculations on models with terminal CO ligands on Fe2 and Fe6 of the FeMo-cofactor. An S=0 model with both CO ligands in exo positions predicts symmetric and asymmetric stretches at equation image=1938 and 1909 cm−1, respectively, with relative band intensities of about 3.5:1, which is in good agreement with experiment. From the observed IR intensities, Hi-3 was found to be present at a concentration about equal to that of the EPR-active Hi-1 species. The relevance of Hi-3 to the nitrogenase catalytic mechanism and its recently discovered Fischer–Tropsch chemistry is discussed.

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