Genetic evidence for coenzyme Q requirement in plasma membrane electron transport.
Genetic evidence for coenzyme Q requirement in plasma membrane electron transport.
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质膜电子传递中辅酶 Q 需求的遗传证据。
DOI：
10.1023/a:1020542230308
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发表时间：
1998
影响因子：
3
通讯作者：
Navas,P
中科院分区：
文献类型：
--
作者：
Santos-Ocaña,C;Villalba,JM;Córdoba,F;Padilla,S;Crane,FL;Clarke,CF;Navas,P
从野生型酿酒酵母粗膜组分中分离的质膜使用多种电子受体（如铁氰化物、细胞色素和抗坏血酸自由基）催化NADH氧化。辅酶Q（泛醌）生物合成缺陷的缺失突变株coq 3 Δ的质膜完全缺乏辅酶Q6，并且含有大大降低水平的NADH-抗坏血酸自由基还原酶活性（约为野生型酵母的10%）。相反，这些膜中缺乏辅酶Q6只导致铁氰化物或细胞色素还原酶的部分抑制。辅酶Q依赖铁氰化物和细胞色素creductases主要是基于超氧化物生成的单电子还原醌半醌。抗坏血酸自由基还原酶是唯一的，因为它高度依赖于辅酶Q，不涉及超氧化物，因为它不受超氧化物歧化酶（SOD）的影响。辅酶Q6和NADH-抗坏血酸自由基还原酶在从用携带野生型COQ 3基因的单拷贝质粒转化的coq 3 Δ菌株获得的质膜和在辅酶Q6存在下生长的coq 3 Δ菌株分离的质膜中被拯救。酶活性被醌拮抗剂氯喹和双香豆素抑制，并在膜溶解后与非变性洗涤剂Zwittergent 3-14。所用的各种抑制剂不影响coq 3 Δ菌株的残余抗坏血酸自由基还原酶。抗坏血酸自由基还原酶在同样呼吸缺陷但在泛醌生物合成中没有缺陷的突变体satp 2 Δ和cor 1 Δ中没有显著改变，表明coq 3 Δ突变体中抗坏血酸自由基还原酶的缺乏仅与不能合成泛醌有关，而与泛醌生物合成缺陷表型无关。对于第一次，我们的研究结果提供了遗传学证据的辅酶Q参与NADH-抗坏血酸自由基还原酶，作为跨膜抗坏血酸稳定的电子源。
Plasma membranes isolated from wild-typeSaccharomyces cerevisiaecrude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochromec, and ascorbate free radical. Plasma membranes from the deletion mutant straincoq3Δ, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6and contained greatly diminished levels of NADH–ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q6in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-creductase. Coenzyme Q dependence of ferricyanide and cytochrome-creductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6and NADH–ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of thecoq3Δ strain with a single-copy plasmid bearing the wild typeCOQ3gene and in plasma membranes isolated form thecoq3Δ strain grown in the presence of coenzyme Q6. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3–14. The various inhibitors used did not affect residual ascorbate free radical reductase of thecoq3Δ strain. Ascorbate free radical reductase was not altered significantly in mutantsatp2Δ andcor1Δ which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase incoq3Δ mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH–ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.