Koutsoupakis Constantinos, Soulimane Tewfik, Varotsis Constantinos
Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece.
J Am Chem Soc. 2003 Dec 3;125(48):14728-32. doi: 10.1021/ja036107e.
The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O(2), NO) requires identification of ligand movement into the binding site and out of the enzyme through specific channels and docking sites. We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba(3) from T. thermophilus, which is responsible for the activation and reduction of both O(2) and NO, to gain insight into the structure of ligand-binding intermediates at ambient temperature. We show that, upon dissociation, the photolyzed CO becomes trapped within a ligand docking site located near the ring A propionate of heme a(3). The 2131 cm(-1) mode of the "docked" CO we have detected corresponds to the B(1) state of Mb and persists for 35 micros. The release of CO from the docking site is not followed by recombination to the heme a(3) Fe. Our analysis indicates that this behavior reflects a mechanism in which the protein near ring A of heme a(3) propionate reorganizes about the released CO from the docking site, and establishes a transient barrier that inhibits the recombination process to the heme a(3) Fe for a few milliseconds. Rebinding to heme a(3) occurs with k(2) = 29.5 s(-1). These results have implications for understanding the role of ligand binding/escape through docking sites and channels in heme-copper oxidases and, thus, in respiration.
对负责激活和催化小分子（O₂、NO）的酶中反应调节的描述，需要确定配体通过特定通道和对接位点进入结合位点并离开酶的过程。我们利用时间分辨步进扫描傅里叶变换红外光谱技术，对嗜热栖热菌的CO光解细胞色素c氧化酶ba₃进行了研究，该酶负责O₂和NO的激活与还原，以深入了解室温下配体结合中间体的结构。我们发现，解离后，光解的CO被困在位于血红素a₃的A环丙酸酯附近的配体对接位点内。我们检测到的“对接”CO的2131 cm⁻¹模式对应于肌红蛋白的B₁状态，并持续35微秒。CO从对接位点释放后，并未与血红素a₃的铁重新结合。我们的分析表明，这种行为反映了一种机制，即血红素a₃丙酸酯A环附近的蛋白质围绕从对接位点释放的CO重新组织，并建立一个短暂的屏障，在几毫秒内抑制与血红素a₃铁的重新结合过程。与血红素a₃的重新结合以k₂ = 29.5 s⁻¹的速率发生。这些结果对于理解血红素 - 铜氧化酶中通过对接位点和通道进行的配体结合/逃逸的作用具有重要意义，从而对于呼吸作用也具有重要意义。
