The striking difference,

however, between FeS and the typical thioredoxin reductases is the absence of the catalytic site with the consensus, Cys-Ala-Thr-Cys-Asp (Fig. 1). As mentioned above, FeS shares 89% identity to the thioredoxin reductase-like protein (PDB ID: 2ZBW) from T. thermophilus HB8. The typical thioredoxin reductase from T. scotoductus SA-01 shares 69% identity with a thioredoxin reductase, for which the structure has also been solved (PDB ID: 2Q7V) (Obiero et al., 2006) from Deinococcus radiodurans. Both these structures are composed of an NAD- as well as an FAD-binding domain connected with an antiparallel β-sheet. Also noteworthy is the secondary structure similarity with regard to α-helices as well as β-sheets present in these two proteins. It has previously been shown that the thioredoxin reductase from E. coli undergoes a large rotational

conformation CHIR-99021 ic50 change between two productive modes – firstly, for electron transfer from NADPH to FAD, and secondly, reduction of the disulphide bond between the redox-active cysteines by FAD (Lennon et al., 2000). isocitrate dehydrogenase inhibitor review This conformational change is thus essential for activity in thioredoxin reductases. Although the ferric reductase reported here has similar structural features compared with prokaryotic thioredoxin reductases, it is unknown whether it will undergo similar conformational changes. The gene encoding the typical thioredoxin reductase was located

in the draft genome sequence of T. scotoductus SA-01 and the translated protein sequence conformed to that typical of thioredoxin reductases as it possesses the redox-active motif known to be responsible for the final transfer of the reducing power to thioredoxin. The FeS and TrxB genes encode proteins with 335 and 325 amino acid residues and Mirabegron predicted molecular masses of 36 147 and 35 132 Da, respectively. Good expressions of both heterologous proteins were obtained and the two-step purification procedure yielded homogenous protein preparations (Fig. 2) at sufficient concentrations for kinetic analysis. The two enzymes were analysed for their ability to reduce ferric iron (Fig. 3). It has previously been shown that flavin reductases are capable of the indirect reduction of ferric iron complexes (Coves & Fontecave, 1993; Woodmansee & Imlay, 2002). Others have also shown the reduction of ferric complexes by enzymes possessing bound flavin, including lipoyl dehydrogenase, NADPH-glutathione reductase, NADH-cytochrome c and NADPH-cytochrome P450 (Petrat et al., 2003). Considering the low redox potential of the FADH2/FAD couple (−0.219 V, E0 at pH 7) and the high redox potentials of most ferric complexes (Pierre et al., 2002), it is not surprising that flavoenzymes are capable of effective ferric reduction.