Fig. 5.

Specific activities of the EtfAB/Bcd variants. The specific activities (µmol substrate oxidized min⁻¹ mg⁻¹ protein) are plotted in red for the NADH bifurcation assay, in orange for the NADH oxidase reaction and in green for the butyryl-CoA dehydrogenase reaction with ferricenium. The enzyme variants can be subdivided into three groups. The first group includes mutations at the Bcd1/Bcd2-domain II interface formed in the D state. Salt bridges are found between Bcd E198−EtfA R243 and Bcd D345−EtfA R243 (see Fig. 3). The second group of mutations addresses the bendable covalent linkers between domain III and the EtfB arm (EtfB F233 and V237) and between domains I and II (EtfA R255). The third group comprises residues that influence the pathway of domain II rotation. EtfB I125 and E165 are located in loop regions contacting domain II. T18 and P40 belong to the EtfB protrusion