Saribaş A S, Ding H, Dutton P L, Daldal F
Department of Biology, Johnson Research Foundation, University of Pennsylvania, Philadelphia 19104.
Biochemistry. 1995 Dec 12;34(49):16004-12. doi: 10.1021/bi00049a014.
In Rhodobacter capsulatus, tyrosine (Y) 147 is a highly conserved residue of the cyt b subunit of the bc1 complex. It is located in the vicinity of residues altered in spontaneous inhibitor resistant mutants that affect the ubihydroquinone oxidase (Qo) site of this enzyme. In this work, Y147 was substituted with phenylalanine (F), valine (V), serine (S), and alanine (A) using site-directed mutagenesis in an effort to investigate its specific role in the Qo site. Of the four mutants obtained, Y147S and Y147A exhibited very low ubihydroquinone:cyt c reductase activities and were unable to support photosynthetic growth (Ps) while Y147F and Y147V were Ps+. In all mutants, no changes in the redox midpoint potentials (Em7) of the cyt bH and cyt bL, the occupancy of the Qo site by Q/QH2, and the flash-induced reverse electron transfer kinetics from Qi to cyt bH were observed. On the other hand, rates of electron transfer from Qo to cyt bH were mildly reduced (2-3-fold) in Y147F and V but dramatically decreased (about 20-fold) in Y147A and S, localizing the defect to the Qo site. Thus, Y147A and S are members of a novel class of Qo site mutants that affect the Qo site catalysis without perturbing the accessibility or binding of the substrate. Additional insight to the role of Y147 on ubihydroquinone oxidation was gained by analyzing the Ps+ revertants of these mutants. Two pseudorevertants contained a second mutation [isoleucine (I) or valine (V)] at the highly conserved M154 position, six residues away from Y147.(ABSTRACT TRUNCATED AT 250 WORDS)
在荚膜红细菌中，酪氨酸（Y）147是bc1复合体cyt b亚基的一个高度保守残基。它位于自发抗抑制剂突变体中发生改变的残基附近，这些突变体影响该酶的泛醌氧化酶（Qo）位点。在这项工作中，利用定点诱变将Y147分别替换为苯丙氨酸（F）、缬氨酸（V）、丝氨酸（S）和丙氨酸（A），以研究其在Qo位点中的具体作用。在获得的四个突变体中，Y147S和Y147A表现出非常低的泛醌：细胞色素c还原酶活性，并且无法支持光合生长（Ps），而Y147F和Y147V是光合生长阳性。在所有突变体中，未观察到cyt bH和cyt bL的氧化还原中点电位（Em7）、Q/QH2对Qo位点的占据以及闪光诱导的从Qi到cyt bH的反向电子转移动力学发生变化。另一方面，Y147F和V中从Qo到cyt bH的电子转移速率略有降低（2至3倍），而Y147A和S中则显著降低（约20倍），表明缺陷位于Qo位点。因此，Y147A和S是一类新型Qo位点突变体的成员，它们影响Qo位点催化而不干扰底物的可及性或结合。通过分析这些突变体的光合生长阳性回复体，对Y147在泛醌氧化中的作用有了更多了解。两个假回复体在高度保守的M154位置（距Y147六个残基）含有第二个突变[异亮氨酸（I）或缬氨酸（V）]。
