ONE-DIMENSIONAL AND 2-DIMENSIONAL ESEEM SPECTROSCOPY OF FLAVOPROTEINS

One-and two-dimensional (ID and 2D) electron spin echo envelope modula tion (ESEEM) spectroscopy was applied to study the flavin cofactors in the neutral semiquinone states of flavodoxin and ferredoxin-NADP(+) r eductase (FNR) from the cyanobacterium Anabaena PCC 7119, and the anio nic semiquinone state of cholesterol oxidase from Brevibacterium stero licum. High-resolution crystal structures are available for all these proteins. Three-and 4-pulse ESEEM and hyperfine sublevel con-elation s pectroscopy (HYSCORE) techniques at X-band were used. HYSCORE spectra showed correlations between transitions caused by interaction of the i soalloxazine unpaired electronic spin present in the semiquinone state with several nitrogen and hydrogen nuclei. Measurements of isotopic l abeled samples ([N-15]FMN flavodoxin and [H-2]flavodoxin) allowed the assignment of ail the detected transitions to nuclei belonging to the FMN cofactor group, Interactions of nitrogens in positions I and 3 of the isoalloxazine ring were determined to have isotropic hyperfine cou pling constants in the 1-2 and 0.5-1 MHz ranges for all the different flavoprotein semiquinones studied, Information about the quadrupolar t erm of these nuclei was also obtained. An intense correlation in the n egative quadrant was detected. It has been , associated to the strongl y interacting N(IO) nucleus. The complete hyperfine term parameters (i ncluding the sign) were obtained from detailed analysis of this signal , being the quadrupolar parameter K, also estimated, Another correlati on in the HYSCORE spectra, corresponding to hydrogen bound to the N(5) position in neutral flavin semiquinones, was detected. Its interactio n parameters were also determined. This study demonstrates that ESEEM spectroscopy, and in particular the HYSCORE technique, are of particul ar utility for detecting and assigning nuclear transition frequencies in flavoprotein semiquinones. Moreover, the results reported here are complementary to ENDOR studies, and both techniques together provide a n import-ant tool for obtaining information about spin distribution in the flavin ring of flavoproteins in the semiquinone slate.