One- and two-dimensional ESEEM spectroscopy of flavoproteins

One- and two-dimensional (1D and 2D) electron spin echo envelope modulation (ESEEM) spectroscopy was applied to study the flavin cofactors in the neutral semiquinone states of flavodoxin and ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119, and the anionic semiquinone state of cholesterol oxidase from Brevibacterium sterolicum. High-resolution crystal structures are available for all these proteins. Three- and 4-pulse ESEEM and hyperfine sublevel correlation spectroscopy (HYSCORE) techniques at X-band were used. HYSCORE spectra showed correlations between transitions caused by interaction of the isoalloxazine unpaired electronic spin present in the semiquinone state with several nitrogen and hydrogen nuclei. Measurements of isotopic labeled samples ([15N]FMN flavodoxin and [2H]flavodoxin) allowed the assignment of all the detected transitions to nuclei belonging to the FMN cofactor group. Interactions of nitrogens in positions 1 and 3 of the isoalloxazine ring were determined to have isotropic hyperfine coupling constants in the 1-2 and 0.5-1 MHz ranges for all the different flavoprotein semiquinones studied. Information about the quadrupolar term of these nuclei was also obtained. An intense correlation in the negative quadrant was detected. It has been associated to the strongly interacting N(10) nucleus. The complete hyperfine term parameters (including the sign) were obtained from detailed analysis of this signal, being the quadrupolar parameter, K, also estimated. Another correlation in the HYSCORE spectra, corresponding to hydrogen bound to the N(5) position in neutral flavin semiquinones, was detected. Its interaction parameters were also determined. This study demonstrates that ESEEM spectroscopy, and in particular the HYSCORE technique, are of particular 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 an important tool for obtaining information about spin distribution in the flavin ring of flavoproteins in the semiquinone state.