Kinetic analysis of F 420 ‐dependent NADP + oxidoreductase (Fno) variants reveal interrupted inner subunit communication

Abstract

F420-dependent NADP+ oxidoreductase (Fno) from Archaeoglobus fulgidus catalyzes the reversible reduction of NADP+ to NADPH within the cell. The catalyzed reaction involves the transfer of a hydride from the C-5 position of the reduced F420 cofactor to the C4 position of NADP+, producing NADPH (Figure 1). Our previous studies on wild-type Fno has determined that the enzyme participates in negative cooperativity and half-site reactivity, suggesting that Fno is a regulatory enzyme and capable of inner subunit communication. Several amino acids have been identified at the interface of the two subunits that are believed to aid in subunit communication. These residues include, R186, H133, T192, T09 and S190. We have created a library of Fno variants to probe the role of these amino acids in subunit communication. We have conducted fluorescence binding experiments, as well as steady-state and pre steady-state kinetic analysis of these Fno variants. Our results have shown that R186, S190 and T192 are involved in subunit communication, switching from negative cooperativity to no cooperativity behavior, unlike H133. Our results are reported here.