7 μmol min-1 mg-1), i.e. showed activity similar to that of quinone: LY3023414 cytochrome c oxidoreductase, while isolated cytochrome oa 3 did not oxidize menaquinol. Interestingly, after adding the fractions containing cytochrome c 553 to cytochrome oa 3 oxidase, TMPD oxidase activity increased ~ 5.0-fold (132 μmol min-1 mg-1 vs 664 μmol min-1 mg-1). Discussion In this study, we isolated a membrane bound cytochrome c 553 from the strictly aerobic hyperthermophilic archaeon, A. pernix. SDS-PAGE analysis

showed 3 bands at apparent molecular masses of 40, 30, and 25 kDa (Figure 4a, panel 1). The measured molecular mass of the 25-kDa band, which was positive for heme staining, was close to the calculated molecular mass for the hypothetical cytochrome BMN 673 chemical structure c subunit encoded by ORF APE_1719.1 (Figure 5). Cytochrome c 553 preparations contained heme B and heme C (Figure 2b, solid line) and catalyzed electron transfer from menaquinone to yeast cytochrome c. On the basis of these results, we concluded that cytochrome c 553 was part of the cytochrome bc complex and that the 3 bands identified by SDS-PAGE analysis corresponded to cytochrome b, Rieske/FeS, and cytochrome c subunits. Data from BN-PAGE analysis supported the idea that these 3 bands are part of the bc complex (Figure 4a, panel 3). The gene for the cytochrome c polypetide, APE_1719.1 contains a CXXCHXnM motif but does not show

high sequence similarity to cytochrome c 1 or the other classes of bacterial or eukaryotic c -type components. It is generally difficult to isolate bc complexes check details from membranes because of their general instability, but the heat stability of this enzyme probably permitted its isolation in this study. We also isolated

a cytochrome oa 3-type cytochrome c oxidase from A. pernix membranes. Based on polypeptide sizes, the upper 2 bands identified by SDS-PAGE (Figure 4b, panel 1) probably corresponded to AoxA (subunit I + III) and AoxB (subunit II). Thus, Sunitinib the partially purified cytochrome oa 3 oxidase here is likely the A-type oxidase identified by Ishikawa et al. previously [10]. Interestingly, cytochrome oa 3 oxidase comigrated with the bc complex through the DEAE-Toyopearl and Q-Sepharose chromatographies, but the enzymes were separated during the subsequent hydroxyapatite chromatography (Figs. S1 and S2). Furthermore, peak fractions from the Q-Sepharose column, which included the bc complex and cytochrome oa 3 oxidase, had menaquinol oxidase activity. These findings suggest that cytochrome oa 3 oxidase forms a supercomplex with the bc complex as observed in some species, such as thermophilic Bacillus PS3 [21], Corynebacterium glutamicum [22], and S. acidocaldarius [15, 23]. Conclusions Here, we showed that A. pernix has a bc complex which includes a c -type cytochrome, and that the bc complex forms supercomplex with the cytochrome oa 3 oxidase.