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Biophys J, September 1999, p. 1712-1720, Vol. 77, No. 3

Electron-Nuclear Double Resonance and Hyperfine Sublevel Correlation Spectroscopic Studies of Flavodoxin Mutants from Anabaena sp. PCC 7119

Milagros Medina,* Anabel Lostao,* Javier Sancho,* Carlos Gómez-Moreno,* Richard Cammack,# Pablo J. Alonso,§ and Jesús I. Martínez§

 *Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009-Zaragoza, Spain;  #Centre for the Study of Metals in Biology and Medicine, Division of Life Sciences, King's College, London W8 7AH, England; and  §Instituto de Ciencia de Materiales de Aragón, Consejo Superior de Investigaciones Científicas-Universidad de Zaragoza, 50009-Zaragoza, Spain

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp57 and Tyr94 at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp57 right-arrow Ala and Tyr94 right-arrow Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.

Biophys J, September 1999, p. 1712-1720, Vol. 77, No. 3
© 1999 by the Biophysical Society   0006-3495/99/09/1712/09  $2.00


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J. L. Casaus, J. A. Navarro, M. Hervas, A. Lostao, M. A. De la Rosa, C. Gomez-Moreno, J. Sancho, and M. Medina
Anabaena sp. PCC 7119 Flavodoxin as Electron Carrier from Photosystem I to Ferredoxin-NADP+ Reductase. ROLE OF TRP57 AND TYR94
J. Biol. Chem., June 14, 2002; 277(25): 22338 - 22344.
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