Expression, purification and crystal structure determination of a ferredoxin reductase from the actinobacterium Thermobifida fusca

Abstract

Ferredoxin reductase FdR9 from Thermobifida fusca, a member of the oxygenase-coupled NADH-dependent ferredoxin reductase (FNR) family, catalyzes electron transfer from NADH to its physiological electron acceptor ferredoxin. It forms part of a three-component cytochrome P450 monooxygenase system in T. fusca. Here, FdR9 was overexpressed and purified and its crystal structure was determined at 1.8 Å resolution. The overall structure of FdR9 is similar to other members of the FNR family and is composed of an FAD-binding domain, an NAD-binding domain and a C-terminal domain. Activity measurements with FdR9 confirmed a strong preference for NADH as the cofactor. Comparison of the FAD- and NAD-binding domains of FdR9 with other ferredoxin reductases revealed the presence of conserved sequence motifs in the FAD-binding domain as well as several highly conserved residues involved in FAD and NAD cofactor binding. Moreover, the NAD-binding site of FdR9 contains a modified Rossmann fold motif, GxSxxS, instead of the classical GxGxxG motif.