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Protein Science (2005), 14:2284-2295. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: Structural determinants of redox potential

Sharmini Alagaratnam1, Gertie van Pouderoyen2, Tjaard Pijning2, Bauke W. Dijkstra2, Davide Cavazzini3, Gian Luigi Rossi3, Walter M.A.M. Van Dongen4, Carlo P.M. van Mierlo4, Willem J. H. van Berkel4 and Gerard W. Canters1

1 Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands
2 Laboratory of Biophysical Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands
3 Department of Biochemistry and Molecular Biology, University of Parma, Italy
4 Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, 6703 HA Wageningen, The Netherlands

(RECEIVED May 10, 2005; FINAL REVISION June 22, 2005; ACCEPTED June 23, 2005)

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel {beta}-sheet flanked on either side by {alpha}-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 310 helix preceding the start of the {alpha}3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.

Keywords: flavodoxin; FMN; hydrogen bonding; polarity; redox potentials

Abbreviations: FMN, flavin mononucleotide • RMS, root mean square • ox, oxidized • sq, semiquinone • hq, hydroquinone.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051582605.

Reprint requests to: Gerard W. Canters, Leiden Institute of Chemistry, P.O. Box 9502, 2300 RA Leiden, The Netherlands; e-mail: canters{at}chem.leidenuniv.nl; fax: +31-71-527-4349.


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