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A substrate channel in the nitrogenase MoFe protein

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JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

Nitrogenase catalyzes the six electron/six proton reduction of N2 to two ammonia molecules at a complex organometallocluster called “FeMo cofactor.” This cofactor is buried within the α-subunit of the MoFe protein, with no obvious access for substrates. Examination of high-resolution X-ray crystal structures of MoFe proteins from several organisms has revealed the existence of a water-filled channel that extends from the solvent-exposed surface to a specific face of FeMo cofactor. This channel could provide a pathway for substrate and product access to the active site. In the present work, we examine this possibility by substituting four different amino acids that line the channel with other residues and analyze the impact of these substitutions on substrate reduction kinetic parameters. Each of the MoFe protein variants was purified and kinetic parameters were established for the reduction of the substrates N2, acetylene, azide, and propyne. For each MoFe protein,Vmax values for the different substrates were found to be nearly unchanged when compared with the values for the wild-type MoFe protein, indicating that electron delivery to the active site is not compromised by the various substitutions. In contrast, theKm values for these substrates were found to increase significantly (up to 22-fold) in some of the MoFe protein variants compared with the wild-type MoFe protein values. Given that each of the amino acids that were substituted is remote from the active site, these results are consistent with the water-filled channel functioning as a substrate channel in the MoFe protein.

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Acknowledgment

This work was supported by a grant from the National Institutes of Health (GM59087).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, UT, 84322, USA

    Brett M. Barney, Michael G. Yurth & Lance C. Seefeldt

  2. Department of Chemistry, Wake Forest University, Winston-Salem, NC, 27109, USA

    Patricia C. Dos Santos

  3. Department of Biochemistry, Fralin Biotechnology Center, Virginia Tech, Blacksburg, VA, 24062, USA

    Dennis R. Dean

Authors
  1. Brett M. Barney
  2. Michael G. Yurth
  3. Patricia C. Dos Santos
  4. Dennis R. Dean
  5. Lance C. Seefeldt

Corresponding authors

Correspondence toDennis R. Dean orLance C. Seefeldt.

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Barney, B.M., Yurth, M.G., Dos Santos, P.C.et al. A substrate channel in the nitrogenase MoFe protein.J Biol Inorg Chem14, 1015–1022 (2009). https://doi.org/10.1007/s00775-009-0544-2

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