Abe Masato, Kubo Akina, Yamamoto Shuhei, Hatoh Yoshinori, Murai Masatoshi, Hattori Yasunao, Makabe Hidefumi, Nishioka Takaaki, Miyoshi Hideto
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Biochemistry. 2008 Jun 10;47(23):6260-6. doi: 10.1021/bi800506s. Epub 2008 May 14.
Studies of the action mechanism of acetogenins, the most potent and structurally unique inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), are valuable in characterizing the inhibitor binding site in this enzyme. Our previous study deepened our understanding of the dynamic function of the spacer region of bis-THF acetogenins [Abe, M., et al. (2005) Biochemistry 44, 14898-14906] but, at the same time, posed new important questions. First, while the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings) span a distance shorter than that of the extended 13 carbon atoms [-(CH 2) 13-], what is the apparent optimal length of the spacer for the inhibition of 13 carbon atoms? In other words, what is the functional role of the additional methylene groups? Second, why was the inhibitory potency of the mono-THF derivative, but not the bis-THF derivative, drastically reduced by hardening the spacer covering 10 carbon atoms into a rodlike shape [-CH 2-(C identical withC) 4-CH 2-]? This study was designed not only to answer these questions but also to further disclose the dynamic functions of the spacer. We here synthesized systematically designed acetogenins, including mono- and bis-THF derivatives, and evaluated their inhibitory effects on bovine complex I. With regard to the first question, we demonstrated that the additional methylenes enhance the hydrophobicity of the spacer region, which may be thermodynamically advantageous for bringing the polar gamma-lactone ring into the membrane-embedded segment of complex I. With regard to the second question, we observed that a decrease in the flexibility of the spacer region is more adverse to the action of the mono-THF series than that of the bis-THF series. As a cause of this difference, we suggest that for bis-THF derivatives, one of the two THF rings, being adjacent to the spacer, is capable of working as a pseudospacer to overcome the remarkable decrease in the conformational freedom and/or the length of the spacer. Moreover, using photoresponsive acetogenins that undergo drastic and reversible conformational changes with alternating UV-vis irradiation, we provided further evidence that the spacer region is free from steric congestion arising from the putative binding site probably because there is no receptor wall for the spacer region.
乙酸原是牛心线粒体复合体I（NADH-泛醌氧化还原酶）最有效且结构独特的抑制剂，对其作用机制的研究有助于确定该酶中抑制剂的结合位点。我们之前的研究加深了对双四氢呋喃乙酸原间隔区动态功能的理解[Abe, M., 等人 (2005) Biochemistry 44, 14898 - 14906]，但同时也提出了新的重要问题。首先，虽然两个毒基团（即羟基化的四氢呋喃和γ-内酯环）跨越的距离比延伸的13个碳原子[-(CH₂)₁₃-]的距离短，但抑制13个碳原子时，间隔区的表观最佳长度是多少？换句话说，额外的亚甲基基团有什么功能作用？其次，为什么将覆盖10个碳原子的间隔区硬化成棒状[-CH₂-(C≡C)₄-CH₂-]会使单四氢呋喃衍生物而非双四氢呋喃衍生物的抑制效力大幅降低？本研究不仅旨在回答这些问题，还旨在进一步揭示间隔区的动态功能。我们在此系统合成了包括单四氢呋喃和双四氢呋喃衍生物在内的经过系统设计的乙酸原，并评估了它们对牛复合体I的抑制作用。关于第一个问题，我们证明额外的亚甲基增强了间隔区的疏水性，这在热力学上可能有利于将极性γ-内酯环带入复合体I的膜嵌入段。关于第二个问题，我们观察到间隔区灵活性的降低对单四氢呋喃系列作用的不利影响比对双四氢呋喃系列更大。作为这种差异的一个原因，我们认为对于双四氢呋喃衍生物，与间隔区相邻的两个四氢呋喃环之一能够作为假间隔区发挥作用，以克服间隔区构象自由度和/或长度的显著降低。此外，使用在交替紫外-可见光照射下会发生剧烈且可逆构象变化的光响应性乙酸原，我们进一步证明间隔区可能没有来自假定结合位点的空间拥挤，这可能是因为间隔区没有受体壁。
