Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix

Abstract

Background: The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species.

Results: Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectrum of the isolated c-type cytochrome showed a characteristic α-band peak at 553 nm corresponding to heme C. The pyridine hemochrome spectrum also revealed the presence of heme B. In non-denaturing polyacrylamide gel electrophoresis, the cytochrome migrated as a single band with an apparent molecular mass of 80 kDa, and successive SDS-PAGE separated the 80-kDa band into 3 polypeptides with apparent molecular masses of 40, 30, and 25 kDa. The results of mass spectrometry indicated that the 25-kDa band corresponded to the hypothetical cytochrome c subunit encoded by the ORF APE_1719.1. In addition, the c-type cytochrome-containing polypeptide complex exhibited menaquinone: yeast cytochrome c oxidoreductase activities.

Conclusion: In conclusion, we showed that A. pernix, a hyperthemophilic archaeon, has a "full" bc complex that includes a c-type cytochrome, and to the best of our knowledge, A. pernix is the first archaea from which such a bc complex has been identified. However, an electron donor candidates for cytochrome c oxidase, such as a blue copper protein, have not yet been identified in the whole genome data of this archaeon. We are currently trying to identify an authentic substrate between a bc complex and terminal oxidase.

Figure 1

Schematic representation of the respiratory chain of Aeropyrum pernix K1. Genes encoding cytochrome c oxidase and other respiratory components in the bacterium are indicated. ORFs APE_1719.1, APE_1724.1 and APE_1725 encode the cytochrome c₅₅₃ complex which was isolated in this study. ORFs APE_0792.1, APE_0793.1 and APE_0795.1, annotated as aoxABC genes, encode an A-type cytochrome c oxidase, and ORFs APE_1623 and APE_1720 encode a B-type cytochrome c oxidase. In the previous study of Ishikawa et al. (2002), these 2 terminal oxidases were designated as cytochrome aa₃- and ba₃-type cytochrome c oxidase, respectively.

Figure 2

Spectra of cytochromes in A. pernix. Difference spectrum in the sodium dithionite-reduced form minus the air-oxidized form (dotted line) and pyridine ferro-hemochromes (solid line) of membranes (a), cytochrome c₅₅₃ (b), and cytochrome oa₃ oxidase (c). To measure a spectrum of membranes, they were solubilized with 5% (w/v) Triton X-100, as described in Materials and Methods. Difference spectrum of the CO-reduced minus the reduced forms of cytochrome oa₃ oxidase (d). The partially purified oxidase was reduced with sodium dithionite (baseline) and then bubbled with CO gas for 1 min.

Figure 3

Heme analysis by MALDI-TOF mass spectrometry of partially purified cytochrome oa₃ oxidase from A. pernix. Heme was extracted from the oxidase preparation by shaking vigorously with acetone-HCl, followed by extraction with ethyl acetate. The extracted heme was analyzed by MALDI-TOF mass spectrometry as detailed in the "Materials and Methods".

Figure 4

SDS-PAGE (panel 1 and 2) and Two-dimensional electrophoresis analysis (panel 3) of the cytochrome c₅₅₃ (a) and cyothcrome oa₃ oxidase (b) from A. pernix. The acrylamide concentration of the SDS-PAGE gel was 13.5%. The gel was stained for protein with CBB (panel 1) and for heme with o-toluidine in the presence of H₂O₂ (panel 2). The samples were analyzed by BN-PAGE (horizontal) and then SDS-PAGE (vertical, panel 3). A 5-18% acrylamide gradient gel was used for native PAGE, and the gels were stained with CBB. The cytochrome oa₃ oxidase was revealed by its TMPD oxidation activity (b panel 3). The acrylamide concentration of the second dimension SDS-PAGE gel was 15%, and the gels were stained with CBB. Side bars indicate the molecular mass standards. The arrows indicate the corresponding subunits of the cytochrome c₅₅₃ and cytochrome oa₃ oxidase.

Figure 5

MALDI-TOF mass spectrum of cytochrome c₅₅₃ from A. pernix. Partially purified cytochrome c₅₅₃ was separated by SDS-PAGE (Figure 4a, panel 1), and the 25-kDa band was extracted from the acrylamide gel. Mass spectrum analysis was performed as detailed in the Materials and Methods.