Spectral evidence for cytochrome o in the brown membrane of Halobacterium halobium

The brown membrane domain of Halobacterium halobium is a developmental precursor of the purple membrane and it contains a b-type cytochrome in addition to bacterio-opsin. In this report we provide spectroscopic evidence that the majority of the cytochrome content is a halobacterial cytochrome o. This cytochrome has absorption spectral properties in the oxidized, reduced, and CO liganded states which are characteristic of cytochrome o. The CD spectra show a complex bilobed pattern in the Soret spectral region which reflects the similarity of the heme environment to those of other b-type cytochromes involved in electron transport. We have also demonstrated a positive cooperativity of CO binding which, combined with CD spectral results, suggests two interacting heme moieties per cytochrome o. Size exclusion HPLC of solubilized brown membrane preparations shows a heme b containing protein with an Mr 42,000-46,000. The cytochrome may be easily separated from bacterio-opsin using a hydroxyapatite elution method on Triton X-100 solubilized preparations. The relative ease with which brown membrane disks may be oriented in high optical quality films should make the brown membrane a valuable model system for future spectroscopic investigations of cytochrome o.