Fractions exhibiting QPO activity that eluted with 0.4–0.5 M potassium phosphate were pooled and loaded onto a 1-mL AF-Red-560M column (Tosoh Corp., Tokyo, Japan). QPO did not bind to the column. The flow-through Obeticholic Acid was pooled and concentrated by the addition of PEG6000 (Yamada et al., 2007). QPO activity was measured by a previously described method (Yamada et al., 2007), with slight modification. This activity was measured at 25 °C in a buffer containing 100 mM Tris-HCl (pH, 7.5), 0.1% (w/v) SM-1200 (Nacalai Tesque Inc.), and ubiquinol-1 (20, 50, 70, 100, 200, and 300 μM). Ubiquinone-1 was kindly gifted by Eisai (Tokyo, Japan), and the reduced form (ubiquinol-1) was

prepared by the method described previously (Rieske, 1967). The reaction was initiated by the addition of 80 μM H2O2. Oxidation of ubiquinol-1 was assessed at 278 nm using an extinction coefficient of 10 mM−1 cm−1. The kinetic

parameters were calculated using graphpad prism (Graphpad software, San Diego, CA) and a nonlinear check details least-squares analysis. Redox titrations were performed using a platinum electrode (Radiomater, Copenhagen, Denmark). The titration was carried out at 25 °C in 100 mM Tris-HCl buffer (pH, 7.5) in the presence of several electron mediators as follows: 50 μM ferrocyanide, 10 μM 2-OH-1,4 naphtoquinone, 20 μM phenazine methosulfate, 20 μM phenazine ethosulfate, 20 μM 2,3,5,6-tetramethyl-p-phenylene diamine, and 20 μM duroquinone (Matsushita et al., 1999). The buffer also contains 0.5% SM-1200 to improve the stability of the measurement system. The course of reduction of heme c was recorded at its α-band maximum at 556.6 nm using MultiSpec-1500 (Shimadzu, Kyoto, Japan). Midpoint potentials were calculated using the Nernst equation for three components

(n=1) with unknown redox potentials with igor pro (WaveMetrics, Lake Oswego, OR) and a nonlinear least-squares analysis. Heterogenous expression of cytochrome c increased by the overexpression of ccm genes and the deletion of degP protease, which is one of the major proteases Sirolimus mw in the periplasmic region of E. coli (Brige et al., 2001). In order to obtain active rQPO, we introduced pET101QPO into Keio:JW0157(DE3)/pCCM, a λDE3-lysogenized strain lacking degP protease and harboring the plasmid pCCM that constitutively expresses ccm genes. We tested several production protocols and found that the highest activity of rQPO was obtained in cultures grown without induction of isopropyl thio-β-d-galactoside. Unfortunately, the His-tag that was introduced into the C-terminus of QPO resulted in the production of inactive rQPO. rQPO with a His-tag at the N-terminus was actively expressed, however, this enzyme was highly unstable upon solubilization (not shown). Because membrane-bound enzymes are difficult to handle, we also attempted to express QPO that lacked the single N-terminal transmembrane region in order to obtain a soluble form of rQPO.