Flavodoxin from Anabaena 7120: uniform nitrogen-15 enrichment and hydrogen-1, nitrogen-15, and phosphorus-31 NMR investigations of the flavin mononucleotide binding site in the reduced and oxidized states

Abstract

Interactions between flavin mononucleotide (FMN) and apoprotein have been investigated in the reduced and oxidized states of the flavodoxin isolated from Anabaena 7120 (Mr .apprx. 21000). 1H, 15N, and 31P NMR have been used to characterize the FMN-protein interaction in both redox states. These are compared with those seen in other flavodoxins. Uniformly enriched [15N]flavodoxin (> 95% isotopic purity) was isolated from Anabaena 7120 grown on K15NO3 as the sole nitrogen source. 15N insensitive nucleus enhanced by polarization transfer (INEPT) and nuclear Overhauser effect (NOE) studies of this sample provided information regarding protein structure and dynamics. A 1H-detected 15N experiment allowed the correlation of nitrogen resonances to those of their attached protons. Over 90% of the expected N-H cross peaks could be resolved in this experiment.