Theissen Ursula, Martin William
Institute of Botany III, Heinrich-Heine University of Duesseldorf, Universitaetsstrasse 1, Duesseldorf, Germany.
FEBS J. 2008 Mar;275(6):1131-9. doi: 10.1111/j.1742-4658.2008.06273.x. Epub 2008 Feb 1.
The lugworm Arenicola marina inhabits marine sediments in which sulfide concentrations can reach up to 2 mM. Although sulfide is a potent toxin for humans and most animals, because it inhibits mitochondrial cytochrome c oxidase at micromolar concentrations, A. marina can use electrons from sulfide for mitochondrial ATP production. In bacteria, electron transfer from sulfide to quinone is catalyzed by the membrane-bound flavoprotein sulfide : quinone oxidoreductase (SQR). A cDNA from A. marina was isolated and expressed in Saccharomyces cerevisiae, which lacks endogenous SQR. The heterologous enzyme was active in mitochondrial membranes. After affinity purification, Arenicola SQR isolated from yeast mitochondria reduced decyl-ubiquinone (K(m) = 6.4 microm) after the addition of sulfide (K(m) = 23 microm) only in the presence of cyanide (K(m) = 2.6 mM). The end product of the reaction was thiocyanate. When cyanide was substituted by Escherichia coli thioredoxin and sulfite, SQR exhibited one-tenth of the cyanide-dependent activity. Six amino acids known to be essential for bacterial SQR were exchanged by site-directed mutagenesis. None of the mutant enzymes was active after expression in yeast, implicating these amino acids in the catalytic mechanism of the eukaryotic enzyme.
沙蠋（Arenicola marina）栖息于海洋沉积物中，其中硫化物浓度可达2 mM。尽管硫化物对人类和大多数动物来说是一种强效毒素，因为它在微摩尔浓度下就能抑制线粒体细胞色素c氧化酶，但沙蠋能够利用硫化物中的电子进行线粒体ATP的生成。在细菌中，从硫化物到醌的电子转移由膜结合黄素蛋白硫化物：醌氧化还原酶（SQR）催化。从沙蠋中分离出一个cDNA，并在缺乏内源性SQR的酿酒酵母中表达。这种异源酶在线粒体内膜中具有活性。经过亲和纯化后，从酵母线粒体中分离出的沙蠋SQR仅在氰化物（Km = 2.6 mM）存在的情况下，加入硫化物（Km = 23 μM）后能还原癸基泛醌（Km = 6.4 μM）。反应的终产物是硫氰酸盐。当用大肠杆菌硫氧还蛋白和亚硫酸盐替代氰化物时，SQR表现出氰化物依赖性活性的十分之一。通过定点诱变替换了已知对细菌SQR至关重要的六个氨基酸。在酵母中表达后，没有一种突变酶具有活性，这表明这些氨基酸参与了真核酶的催化机制。
