FIG 4.

(A) SDS-PAGE analysis of the purified recombinant Euo. M, protein marker; 1, purified protein. (B) Changes in UV-visible absorption spectra during the reduction of CoQ₁ with reduced EtfAB-I by Euo coupling with the reduction of EtfAB-I with pseudooxynicotine by Pno. Initially, the reaction mixture contained 50 mM Tris-HCl (pH 8.5), 1 mM pseudooxynicotine, 0.05 μM purified Pno, and 30 μM purified Euo (black line). The mixture was then treated by adding 8 μM purified EtfAB-I (red line). The reduction of Euo proceeded with time (light-blue and dark-green lines). Upon stabilization of the spectrum, 32 μM CoQ₁ was added and Euo was reoxidized (pink line). The reoxidization proceeded with time (light-green and dark-blue lines). The downward arrows indicate the reduction of Euo after the addition of EtfAB-I, whereas the upward arrows indicate the reoxidation of Euo after the addition of CoQ₁. Neither oxidized nor reduced CoQ₁ has absorption in the wavelength window. The time between the first two records (between the black line and dark-blue line) was 45 s. The rest of the records were done every 45 s.