Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

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• 作者：Nikaido ; Keiichi ; Sakamoto ; Junshi ; Noguchi ; Shunsuke ; Sone ; Nobuhito
• 关键词：SoxB-type heme-copper oxidase ; Cytochrome bo₃ ; Respiratory chain ; Cytochrome c-551 ; Proton pump ; (Bacillus stearothermophilus) ; Bst ; Bacillus stearothermophilus ; CCCP ; carbonylcyanide m-chlorophenyl-hydrazone ; MEGA 9+10 ; 1 ; 1 mixture of n-nonano
• 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
• 出版年：2000
• 出版时间：January 3, 2000
• 年：2000
• 卷：1456
• 期：1
• 页码：35-44
• 全文大小：280 K

文摘

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo₃-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo₃-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H⁺ pumping activity, although the efficiency was lower than those of cytochrome aa₃-type oxidases belonging to the SoxM-type.