Hartsel S C, Kolodziej B J, Cassim J Y
Department of Microbiology, Ohio State University, Columbus 43210.
Arch Biochem Biophys. 1988 Jul;264(1):74-81. doi: 10.1016/0003-9861(88)90572-3.
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.
嗜盐菌的棕色膜结构域是紫膜的发育前体，除细菌视紫红质外还含有一种b型细胞色素。在本报告中，我们提供了光谱学证据，表明大部分细胞色素含量是一种嗜盐菌细胞色素o。这种细胞色素在氧化态、还原态和CO配位态下具有吸收光谱特性，这是细胞色素o的特征。圆二色光谱在Soret光谱区域显示出复杂的双叶模式，这反映了血红素环境与参与电子传递的其他b型细胞色素的相似性。我们还证明了CO结合的正协同性，结合圆二色光谱结果，表明每个细胞色素o有两个相互作用的血红素部分。对溶解的棕色膜制剂进行尺寸排阻高效液相色谱显示，有一种含血红素b的蛋白质，其分子量为42,000 - 46,000。使用羟基磷灰石洗脱法对Triton X - 100溶解的制剂进行处理，可以很容易地将细胞色素与细菌视紫红质分离。棕色膜盘在高质量光学薄膜中易于定向，这应该使棕色膜成为未来细胞色素o光谱研究的有价值的模型系统。
