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Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin

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Summary

The 1H, 15N and 13C backbone and 1H and 13C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30°C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, 3JHnHα coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel β-sheet and five α-helices. One of the outer regions of the β-sheet shows no regular extended conformation, whereas the outer strand β4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five α-helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the α-helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn11, Ser68 and Asn72 are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.

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Abbreviations

SQ:

semiquinone

FMN:

riboflavin 5′-monophosphate; nif, nitrogen fixation

TSP:

3-(trimethylsilyl)propionate sodium salt

DSS:

2,2-dimethyl-2-silapentane-5-sulfonate sodium salt

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Authors and Affiliations

  1. Department of Biochemistry, Agricultural University, Dreijenlaan 3, NL-6703 HA, Wageningen, The Netherlands

    Sjaak Peelen, Paul J. A. Erbel & Jacques Vervoort

  2. SON/NWO National HF-NMR Facility, University of Nijmegen, Toernooiveld, NL-6525 ED, Nijmegen, The Netherlands

    Sybren S. Wijmenga

  3. School of Animal and Microbial Sciences, University of Reading, P.O. Box 228, RG6 6AJ, Reading, UK

    Robert L. Robson

  4. Nitrogen Fixation Laboratory, John Innes Centre, NRA 7HH, Norwich, UK

    Robert R. Eady

Authors
  1. Sjaak Peelen
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  2. Sybren S. Wijmenga
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  3. Paul J. A. Erbel
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  4. Robert L. Robson
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  5. Robert R. Eady
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  6. Jacques Vervoort
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Additional information

Supplementary Material is available on request, comprising a Materials and Methods section for the expression and purification of the A. chroococcum flavodoxin, a Table S1 containing the parameters of the titration of A. chroococcum flavodoxin with the Fe protein, and a Table S2 containing the 15N, HN, 13Cα, 1Hα, 13Cβ, 1Hβ and 13CO chemical shifts.

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Peelen, S., Wijmenga, S.S., Erbel, P.J.A. et al. Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin. J Biomol NMR 7, 315–330 (1996). https://doi.org/10.1007/BF00200433

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  • DOI: https://doi.org/10.1007/BF00200433

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