Mather M W, Yu L, Yu C A
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, USA.
J Biol Chem. 1995 Dec 1;270(48):28668-75. doi: 10.1074/jbc.270.48.28668.
The cytochrome b subunit (subunit I) of the ubiquinolcytochrome c reductase (bc1 complex) is thought to participate in the formation of two quinone/quinol reaction centers, an oxidizing center (Qo) and a reducing center, in accordance with the quinone cycle mechanism. Threonine 160 is a highly conserved residue in a segment of subunit I that was shown to bind quinone and is placed near the putative Qo site in current models of the bc1 complex. Rhodobacter sphaeroides cells expressing bc1 complexes with Ser or Tyr substituted for Thr160 grow photosynthetically at a reduced rate, and cells expressing the mutated complexes produce an "elevated" level of the bc1 complex. The Ser substitution also affects the interaction of subunit IV with subunit I. Replacement of Thr160 by Ser results in about a 70% loss of the activity in the purified complex, whereas substitution by Tyr lowers the activity by more than 80%. Both replacements lower the apparent Km for ubiquinol. Electron paramagnetic resonance (EPR) spectroscopy shows that in the Ser substituted complex, the environments of the Rieske iron-sulfur cluster in subunit III and the high potential cytochrome b (b562) in subunit I have been modified. The spectra of the Ser160 and Tyr160 iron-sulfur clusters have become redox-insensitive, with a line shape resembling that of the native complex in the fully reduced state. The EPR signal of b562 in the Ser160 complex is shifted from g = 3.50 to g = 3.52, but otherwise the line shape is very similar to the spectrum of the native complex. Most of these results are consistent with current ideas regarding the structure and function of Qo in the bc1 complex, except for the alteration of the b562 EPR feature, because this heme is not thought to be located in proximity to Qo. Immunoblotting analysis showed that the Ser or Tyr substituted complex contained significantly less than a stoichiometric amount of subunit IV. The enzymatic activity of mutated bc1 complex was found to be activable by the addition of purified subunit IV. These results indicate that Thr160 plays an important role in the structure and/or function of the bc1 complex.
泛醌 - 细胞色素c还原酶（bc1复合物）的细胞色素b亚基（亚基I）被认为根据醌循环机制参与两个醌/醌醇反应中心的形成，即一个氧化中心（Qo）和一个还原中心。苏氨酸160是亚基I一段区域中高度保守的残基，该区域已被证明能结合醌，并且在当前bc1复合物模型中位于假定的Qo位点附近。表达用丝氨酸或酪氨酸取代苏氨酸160的bc1复合物的球形红细菌细胞光合生长速率降低，并且表达突变复合物的细胞产生“升高”水平的bc1复合物。丝氨酸取代也影响亚基IV与亚基I的相互作用。用丝氨酸取代苏氨酸160导致纯化复合物中约70%的活性丧失，而用酪氨酸取代则使活性降低超过80%。两种取代都降低了对泛醌醇的表观Km值。电子顺磁共振（EPR）光谱表明，在丝氨酸取代的复合物中，亚基III中的 Rieske 铁硫簇和亚基I中的高电位细胞色素b（b562）的环境已被改变。丝氨酸160和酪氨酸160铁硫簇的光谱对氧化还原不敏感，其线形类似于完全还原状态下天然复合物的线形。丝氨酸160复合物中b562的EPR信号从g = 3.50 移至g = 3.52，但除此之外，线形与天然复合物的光谱非常相似。除了b562 EPR特征的改变外，这些结果大多与当前关于bc1复合物中Qo的结构和功能的观点一致，因为这个血红素不被认为位于Qo附近。免疫印迹分析表明，丝氨酸或酪氨酸取代的复合物所含亚基IV的量明显少于化学计量。发现添加纯化的亚基IV可激活突变bc1复合物的酶活性。这些结果表明苏氨酸160在bc1复合物的结构和/或功能中起重要作用。
