Fato Romana, Bergamini Christian, Bortolus Marco, Maniero Anna Lisa, Leoni Serena, Ohnishi Tomoko, Lenaz Giorgio
Dipartimento di Biochimica G. Moruzzi, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy.
Biochim Biophys Acta. 2009 May;1787(5):384-92. doi: 10.1016/j.bbabio.2008.11.003. Epub 2008 Nov 14.
We have investigated the production of reactive oxygen species (ROS) by Complex I in isolated open bovine heart submitochondrial membrane fragments during forward electron transfer in presence of NADH, by means of the probe 2',7'-Dichlorodihydrofluorescein diacetate. ROS production by Complex I is strictly related to its inhibited state. Our results indicate that different Complex I inhibitors can be grouped into two classes: Class A inhibitors (Rotenone, Piericidin A and Rolliniastatin 1 and 2) increase ROS production; Class B inhibitors (Stigmatellin, Mucidin, Capsaicin and Coenzyme Q(2)) prevent ROS production also in the presence of Class A inhibitors. Addition of the hydrophilic Coenzyme Q(1) as an electron acceptor potentiates the effect of Rotenone-like inhibitors in increasing ROS production, but has no effect in the presence of Stigmatellin-like inhibitors; the effect is not shared by more hydrophobic quinones such as decyl-ubiquinone. This behaviour relates the prooxidant CoQ(1) activity to a hydrophilic electron escape site. Moreover the two classes of Complex I inhibitors have an opposite effect on the increase of NADH-DCIP reduction induced by short chain quinones: only Class B inhibitors allow this increase, indicating the presence of a Rotenone-sensitive but Stigmatellin-insensitive semiquinone species in the active site of the enzyme. The presence of this semiquinone was also suggested by preliminary EPR data. The results suggest that electron transfer from the iron-sulphur clusters (N2) to Coenzyme Q occurs in two steps gated by two different conformations, the former being sensitive to Rotenone and the latter to Stigmatellin.
我们借助探针2',7'-二氯二氢荧光素二乙酸酯，研究了在NADH存在下，分离的开放型牛心亚线粒体膜片段中，复合物I在正向电子传递过程中活性氧（ROS）的产生情况。复合物I产生ROS与其抑制状态密切相关。我们的结果表明，不同的复合物I抑制剂可分为两类：A类抑制剂（鱼藤酮、杀粉蝶菌素A以及罗利抑素1和2）会增加ROS的产生；B类抑制剂（柱晶白霉素、粘菌素、辣椒素和辅酶Q(2)）即使在存在A类抑制剂的情况下也能阻止ROS的产生。添加亲水性辅酶Q(1)作为电子受体，会增强鱼藤酮类抑制剂增加ROS产生的效果，但在存在柱晶白霉素类抑制剂时则没有效果；更疏水的醌类如癸基泛醌则没有这种作用。这种行为将促氧化的辅酶Q(1)活性与一个亲水性电子逃逸位点联系起来。此外，两类复合物I抑制剂对短链醌类诱导的NADH-DCIP还原增加有相反的作用：只有B类抑制剂能使这种增加发生，这表明在该酶的活性位点存在一种对鱼藤酮敏感但对柱晶白霉素不敏感的半醌物种。初步的电子顺磁共振（EPR）数据也表明了这种半醌的存在。结果表明，从铁硫簇（N2）到辅酶Q的电子传递分两步进行，由两种不同的构象控制，前者对鱼藤酮敏感，后者对柱晶白霉素敏感。
