Electron-nuclear double resonance and hyperfine sublevel correlation spectroscopic studies of flavodoxin mutants from Anabaena sp PCC 7119

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin de nsity distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquin one radicals of the proteins were obtained by photoreduction and examined b y electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlat ion (HYSCORE) spectroscopies. Significant differences in electron density d istribution were observed in the flavodoxin mutants Trp57, Al, and Ty(94) - -> Ala. The results indicate that the presence of a bulky residue (either a romatic or aliphatic) at position 57, as compared with an alanine, decrease s the electron spin density in the nuclei of the benzene flavin ring, where as an aromatic residue at position 94 increases the electron spin density a t positions N(5) and C(6) of the flavin ring. The influence of the FMN ribi tyl and phosphate on the flavin semiquinone was determined by reconstitutin g apoflavodoxin samples with riboflavin and with lumiflavin. The coupling p arameters of the different nuclei of the isoalloxazine group, as detected b y ENDOR and HYSCORE, were very similar to those of the native flavodoxin. T his 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.