Department of Chemistry and Food Chemistry, Technische Universität Dresden, 01069 Dresden, Germany.
Department of Chemistry, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany.
J Am Chem Soc. 2021 Feb 24;143(7):2769-2776. doi: 10.1021/jacs.0c10767. Epub 2021 Feb 9.
Cytochrome oxidase (CcO) in its as-isolated form is known to exist in a slow and fast form, which differ drastically in their ability to bind oxygen and other ligands. While preparation methods have been established that yield either the fast or the slow form of the protein, the underlying structural differences have not been identified yet. Here, we have performed surface enhanced resonance Raman (SERR) spectroscopy of CcO immobilized on electrodes in both forms. SERR spectra obtained in resonance with the heme metal-to-ligand charge transfer (MLCT) transition at 650 nm displayed a sharp vibrational band at 748 or 750 cm when the protein was in its slow or fast form, respectively. DFT calculations identified the band as a mode of the His-419 ligand that is sensitive to the oxygen ligand and the protonation state of Tyr-288 within the binuclear complex. Potential-dependent SERR spectroscopy showed a redox-induced change of this band around 525 mV versus Ag/AgCl exclusively for the fast form, which coincides with the redox potential of the Tyr-O/Tyr-O transition. Our data points to a peroxide ligand in the resting state of CcO for both forms. The observed frequencies and redox sensitivities of the Raman marker band suggest that a radical Tyr-288 is present in the fast form and a protonated Tyr-288 in the slow form.
细胞色素氧化酶（CcO）在其原始形式中存在慢型和快型两种形式，它们在结合氧和其他配体的能力上有很大的差异。虽然已经建立了制备方法，可以得到蛋白质的快型或慢型，但尚未确定其潜在的结构差异。在这里，我们对固定在电极上的两种形式的 CcO 进行了表面增强共振拉曼（SERR）光谱研究。在与血红素金属-配体电荷转移（MLCT）跃迁在 650nm 处共振的 SERR 光谱中，当蛋白质处于慢型或快型时，分别在 748 或 750cm 处显示出一个尖锐的振动带。DFT 计算将该带识别为双核配合物中 His-419 配体的一种模式，该模式对氧配体和 Tyr-288 的质子化状态敏感。电位依赖性 SERR 光谱显示，仅在快型中，该带在 525mV 左右发生与 Ag/AgCl 相比的氧化还原诱导变化，这与 Tyr-O/Tyr-O 转变的氧化还原电位一致。我们的数据表明，两种形式的 CcO 在静止状态下都存在过氧化物配体。观察到的拉曼标记带的频率和氧化还原灵敏度表明，在快型中存在自由基 Tyr-288，而在慢型中存在质子化的 Tyr-288。
