Convenient synthesis of deazaflavin cofactor FO and its activity in F420-dependent NADP reductase

Abstract

F₄₂₀ and FO are phenolic 5-deazaflavin cofactors that complement nicotinamide and flavin redox coenzymes in biochemical oxidoreductases and photocatalytic systems. Specifically, these 5-deazaflavins lack the single electron reactivity with O₂ of riboflavin-derived coenzymes (FMN and FAD), and, in general, have a more negative redox potential than NAD(P)⁺. For example, F₄₂₀-dependent NADP⁺ oxidoreductase (Fno) is critical to the conversion of CO₂ to CH₄ by methanogenic archaea, while FO functions as a light-harvesting agent in DNA repair. The preparation of these cofactors is an obstacle to their use in biochemical studies and biotechnology. Here, a convenient synthesis of FO was achieved by improving the redox stability of synthetic intermediates containing a polar, electron-rich aminophenol fragment. Improved yields and simplified purification techniques for FO are described. Additionally, Fno activity was restored with FO in the absence of F₄₂₀. Investigating the FO-dependent NADP⁺/NADPH redox process by stopped-flow spectrophotometry, steady state kinetics were defined as having a K_m of 4.00 ± 0.39 μM and a k_cat of 5.27 ± 0.14 s⁻¹. The preparation of FO should enable future biochemical studies and novel uses of F₄₂₀ mimics.