Xiao Kunhong, Liu Xiaoying, Yu Chang-An, Yu Linda
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Biochemistry. 2004 Feb 17;43(6):1488-95. doi: 10.1021/bi035378z.
Sequence alignment of the Rieske iron-sulfur protein (ISP) of cytochrome bc(1) complex from various sources reveals that bacterial ISPs contain an extra fragment. To study the role of this fragment in bacterial cytochrome bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with deletion or single- or multiple-alanine substitution at various positions of this fragment (residues 96-107) were generated and characterized. The ISPDelta(96-107), ISP(96-107)A, and ISP(104-107)A mutant cells, in which residues 96-107 of ISP are deleted, and residues 96-107 and 104-107 are substituted with alanine, respectively, do not grow photosynthetically and show no bc(1) complex activity in intracytoplasmic membranes prepared from these mutant cells. The ISP(96-99)A, in which residues 96-99 are substituted with alanine, grows photosynthetically at a rate comparable to that of the complement cells, whereas ISP(100-103)A, in which residues 100-103 are substituted with alanine, has a longer lag period prior to photosynthetic growth. Chromatophores prepared from these two mutant cells have 48% and 9% of the bc(1) activity found in the complement chromatophores. The loss (or decrease) of bc(1) activity in these mutant membranes results from a lack (or decrease) of ISP in the membrane due to ISP protein instability and not from mutations affecting the assembly of cytochromes b and c(1) into the membrane, the binding affinity of cytochrome b to cytochrome c(1), or the ability of these two cytochromes to interact with ISP or subunit IV. The order of essentiality of residues in this fragment is residues 104-107 > residues 100-103 > residues 96-99.
对来自不同来源的细胞色素bc(1)复合物的 Rieske 铁硫蛋白（ISP）进行序列比对发现，细菌的ISP含有一个额外的片段。为了研究该片段在细菌细胞色素bc(1)复合物中的作用，构建并表征了球形红杆菌突变体，这些突变体表达在该片段（第96 - 107位氨基酸）的不同位置带有His标签且存在缺失、单丙氨酸取代或多丙氨酸取代的细胞色素bc(1)复合物。ISP缺失(96 - 107)、ISP(96 - 107)A和ISP(104 - 107)A突变体细胞中，ISP的第96 - 107位氨基酸被删除，以及第96 - 107位和104 - 107位氨基酸分别被丙氨酸取代，这些突变体细胞不能进行光合生长，并且从这些突变体细胞制备的胞内膜中未显示出bc(1)复合物活性。ISP(96 - 99)A中第96 - 99位氨基酸被丙氨酸取代，其光合生长速率与互补细胞相当，而ISP(100 - 103)A中第100 - 103位氨基酸被丙氨酸取代，在光合生长之前有更长的延迟期。从这两个突变体细胞制备的载色体具有互补载色体中bc(1)活性的48%和9%。这些突变膜中bc(1)活性的丧失（或降低）是由于ISP蛋白不稳定导致膜中ISP缺乏（或减少），而不是由于影响细胞色素b和c(1)组装到膜中、细胞色素b与细胞色素c(1)的结合亲和力，或这两种细胞色素与ISP或亚基IV相互作用能力的突变。该片段中氨基酸残基的必需性顺序为第104 - 107位氨基酸>第100 - 103位氨基酸>第96 - 99位氨基酸。
