Matsushita K, Yakushi T, Toyama H, Shinagawa E, Adachi O
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753, Japan.
J Biol Chem. 1996 Mar 1;271(9):4850-7. doi: 10.1074/jbc.271.9.4850.
Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH.
醋酸菌的乙醇脱氢酶（ADH）作为乙醇氧化酶呼吸链的主要脱氢酶，在此过程中它将电子传递给泛醌。ADH是一种膜结合的醌血红蛋白 - 细胞色素c复合物，由亚基I（78 kDa）、II（48 kDa）和III（14 kDa）组成，含有几个血红素c以及吡咯并喹啉醌作为辅基。为了了解血红素c部分在分子内电子传递和泛醌还原中的作用，将氧化葡萄糖杆菌的ADH复合物分离成亚基I/III复合物和亚基II，然后再重组为复合物。亚基I/III复合物，可能是亚基I，各自含有1摩尔的吡咯并喹啉醌和血红素c，并表现出显著的铁氰化物还原酶活性，但没有Q1还原酶活性。亚基II是一种三血红素细胞色素c，没有酶活性，但它能使亚基I/III复合物重现Q1和铁氰化物还原酶活性。构建了由氧化葡萄糖杆菌ADH的亚基I/III复合物和醋酸醋杆菌ADH的亚基II组成的杂合ADH，它表现出显著的Q1还原酶活性，表明亚基II有一个泛醌结合位点。从活性ADH中分别分离出了氧化葡萄糖杆菌的无活性ADH，其Q1和铁氰化物还原酶活性仅为活性酶的10%（松下健、薮志、高木洋、远山浩、安达雄（1995年）《细菌学杂志》177卷，6552 - 6559页）。使用这些活性和无活性的ADH以及分离出的亚基I/III复合物，我们进行了动力学研究，结果表明ADH含有四个与铁氰化物反应的位点，其中一个在亚基I中检测到，其他三个在亚基II中。亚基II中三个与铁氰化物反应的位点之一在无活性ADH中存在缺陷。即使在对无活性ADH进行碱处理后以及从亚基重组ADH复合物后，与铁氰化物反应的位点仍然无活性，这与Q1还原酶活性和其他铁氰化物还原酶活性的恢复形成对比。因此，数据表明亚基I中的血红素c以及亚基II中三个血红素c部分中的两个参与了ADH分子内电子传递到泛醌的过程，其中两个血红素c位点中的一个可能在泛醌反应位点处或其附近起作用，另一个在该位点与亚基I中的血红素c位点之间。亚基II中剩余的血红素c部分可能具有除了在ADH中将电子从乙醇传递到泛醌之外的其他功能。
