Vos M H, Lipowski G, Lambry J C, Martin J L, Liebl U
Laboratory for Optical Biosciences, INSERM U451, CNRS URA 7645, Ecole Polytechnique-ENSTA, 91128 Palaiseau Cedex, France.
Biochemistry. 2001 Jul 3;40(26):7806-11. doi: 10.1021/bi010060x.
Nitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed.
一氧化氮（NO）通过在细胞色素c氧化酶的双核活性位点作为分子氧的竞争性配体参与呼吸调节。该位点及其附近的NO动态尚未完全了解。我们使用飞秒瞬态吸收光谱对反硝化副球菌细胞色素c氧化酶中血红素a3的NO进行了闪光光解研究。产物态（未配位的血红素a3基态）的形成以与先前观察到的该酶一氧化碳光解类似的逐步方式（周期约700飞秒）发生，并根据亚皮秒时间尺度上活性位点中的弹道配体运动进行了解释[Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181 - 184]。解离的NO的一部分（在非常低的NO浓度下约为35%）在200 - 300皮秒内与血红素a3重新结合。这种重新结合阶段的存在表明与第二个配体结合位点（一个铜原子（CuB））的瞬态键寿命很短或可能未形成。增加NO浓度会在数百皮秒的时间尺度上增加重新结合产率。这种效应在血红素蛋白中是前所未有的，这意味着除了一个与血红素a3结合的NO分子外，即使在相对较低（亚微摩尔）浓度下，第二个NO分子也可以容纳在活性位点中。基于分子动力学能量最小化提出了活性位点中NO容纳的模型。讨论了NO运动的途径及其与呼吸调节的相关性。
