Koutsoupakis Konstantinos, Stavrakis Stavros, Pinakoulaki Eftychia, Soulimane Tewfik, Varotsis Constantinos
Department of Chemistry, University of Crete, 71409 Heraklion, Crete, Greece and Paul Scherrer Institut, Life Sciences, OSRA/008, CH-5232 Villigen PSI, Switzerland.
J Biol Chem. 2002 Sep 6;277(36):32860-6. doi: 10.1074/jbc.M204943200. Epub 2002 Jul 3.
We report the first evidence for the existence of the equilibrium Cu(B)1+-CO species of CO-bound reduced cytochrome ba(3) from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu(B)1+-CO is located at 2053 cm(-1) and remains unchanged in H(2)O/D(2)O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu(B) histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu(B)-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient Cu(B)1+-CO complex is 34.5 s(-1) and rebinding to heme a(3) occurs with k(2) = 28.6 s(-1). The rate of decay of the transient Cu(B)1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a(3) propionates (COOH). The nu(C-O) of the transient Cu(B)1+-CO species is the same as that of the equilibrium Cu(B)1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu(B) between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.
我们报道了嗜热栖热菌中与一氧化碳结合的还原型细胞色素ba(3)的平衡态Cu(B)1+-CO物种在室温下存在的首个证据。Cu(B)1+-CO的C-O伸缩振动模式频率位于2053 cm(-1)，在H(2)O/D(2)O交换中以及在pD 5.5至9.7之间保持不变，这表明化学环境不会改变Cu(B)组氨酸配体的质子化状态。此处报道的数据和结论与最近报道的Cu(B)-His-290质子化状态的变化相反（Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039 - 2045以及Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591 - 9596）。时间分辨步进扫描傅里叶变换红外差谱表明，瞬态Cu(B)1+-CO复合物的衰减速率为34.5 s(-1)，以k(2) = 28.6 s(-1)的速率重新结合到血红素a(3)上。瞬态Cu(B)1+-CO复合物的衰减速率显示出与1694(+)/1706(-)处的吸收变化相似的时间常数，这归因于血红素a(3)丙酸酯（COOH）的扰动。瞬态Cu(B)1+-CO物种的ν(C-O)与平衡态Cu(B)1+-CO物种的相同，并且在pD范围5.5 - 9.7内保持不变，这表明在这些状态之间Cu(B)没有发生结构变化。讨论了这些结果对血红素 - 铜氧化酶中质子途径的影响。
