Chen Y R, Usui S, Yu C A, Yu L
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078.
Biochemistry. 1994 Aug 23;33(33):10207-14. doi: 10.1021/bi00199a050.
Rhodobacter sphaeroides mutants lacking subunit IV (M(r) = 14,384) of the cytochrome b-c1 complex (representative mutant strain, RS delta IV-2) have been constructed by site-specific recombination between the wild-type genomic subunit IV structural gene (fbcQ) and a suicide plasmid containing a defective fbcQ sequence. RS delta IV-2 gives rise to a photosynthetically competent phenotype after a period of adaptation. The chemical compositions, spectral properties, and cytochrome b-c1 complex activities in subunit IV-deficient chromatophores from adapted RS delta IV-2 are similar to those in wild-type chromatophores. However, the apparent Km for Q2H2 for the b-c1 complex in subunit IV-deficient chromatophores from adapted RS delta IV-2 cells is about four times higher than that in chromatophores from wild-type cells. The cytochrome b-c1 complex activity in subunit IV-deficient chromatophores of adapted RS delta IV-2 cells is more labile to detergent treatment than that from wild-type cells. The specific activities of dodecylmaltoside-solubilized fractions of RS delta IV-2, based on cytochrome b, are only one-fourth that of the untreated chromatophores. Introducing a wild-type fbcQ operon on a stable low copy number plasmid, pRK415, into RS delta IV-2 restores photosynthetic growth behavior, the apparent Km value for Q2H2, and tolerance to detergent treatment to that of wild-type cells. Cytochrome b-c1 complex purified from adapted RS delta IV-2 contains only three subunits. It has only 25% of the activity of the four-subunit enzyme. This low activity is accompanied by an increase of the apparent Km for Q2H2 from 3 to 13 microM, suggesting that subunit IV may be involved in quinone binding in addition to its structural role.
通过野生型基因组亚基IV结构基因（fbcQ）与含有缺陷fbcQ序列的自杀质粒之间的位点特异性重组，构建了缺乏细胞色素b-c1复合体亚基IV（分子量=14,384）的球形红杆菌突变体（代表性突变菌株RS delta IV-2）。经过一段时间的适应后，RS delta IV-2产生了光合活性表型。适应后的RS delta IV-2中缺乏亚基IV的载色体的化学组成、光谱特性和细胞色素b-c1复合体活性与野生型载色体中的相似。然而，适应后的RS delta IV-2细胞中缺乏亚基IV的载色体中b-c1复合体对Q2H2的表观Km值比野生型细胞载色体中的高约四倍。适应后的RS delta IV-2细胞中缺乏亚基IV的载色体中的细胞色素b-c1复合体活性比野生型细胞的更易受去污剂处理的影响。基于细胞色素b，RS delta IV-2的十二烷基麦芽糖苷溶解组分的比活性仅为未处理载色体的四分之一。将稳定的低拷贝数质粒pRK415上的野生型fbcQ操纵子导入RS delta IV-2，可恢复光合生长行为、对Q2H2的表观Km值以及对去污剂处理的耐受性至野生型细胞水平。从适应后的RS delta IV-2中纯化的细胞色素b-c1复合体仅包含三个亚基。它的活性仅为四亚基酶的25%。这种低活性伴随着Q2H2的表观Km值从3 microM增加到13 microM，表明亚基IV除了其结构作用外，可能还参与醌的结合。
