19F NMR studies with 2'-F-2'-deoxyarabinoflavoproteins

Apoproteins of several flavoproteins were reconstituted with 2'-F-2'-deoxyarabinoflavins and studied by 19F NMR and absorption spectroscopy. Extensive protein-fluorine interactions were observed by large chemical shift changes on binding to the apoprotein of Old Yellow Enzyme (apoOYE) and apoflavodoxin, whereas binding to apoglucose oxidase and apo -amino acid oxidase (apoDAAO) resulted in minimal interactions. Modification at the flavin 2'-position in OYE resulted in a substantial decrease in the binding affinity of the flavin, possibly from the disruption of two important hydrogen bonds to Pro-35 and Arg-243. 19F NMR studies of complexes of OYE with testosterone, cyclohexenone, and beta-estradiol suggest that phenols and alpha,beta-unsaturated ketones orient differently at the active site on binding. The two separate one-electron potentials for the EFlox/EFlsq and EFlsq/EFlred couples were different for the reconstituted OYE. With native enzyme, there is 15-20% thermodynamic stabilization of the anionic flavin semiquinone, while no detectable amount of semiquinone was observed with modified OYE. This change in potential was further substantiated by blue shifts for the maxima of the modified protein-phenol charge transfer complexes. In accordance with the crystal structure of the OYE-p-OH-benzaldehyde complex (Fox, K.M. & Karplus, P.A. (1994) Structure 2, 1089-1105), 19F NMR studies with the modified OYE-2,4-F2-phenol suggest strong interaction between the para-fluorine of the phenol and Tyr-375.