Gautier Clément, Mikula Ivan, Nioche Pierre, Martasek Pavel, Raman C S, Slama-Schwok Anny
CNRS, UMR 7645, Laboratory of Optics and Biosciences, Ecole Polytechnique, 91128 Palaiseau, France.
Nitric Oxide. 2006 Dec;15(4):312-27. doi: 10.1016/j.niox.2006.03.010. Epub 2006 Apr 5.
Some Gram-positive bacterial pathogens harbor a gene that encodes a protein (HNS, Heme domain of NO Synthase-like proteins) with striking sequence identity to the oxygenase domain of mammalian NO synthases (NOS). However, they lack the N-terminal and the Zn-cysteine motif participating to the stability of an active dimer in the mammalian isoforms. The unique properties of HNS make it an excellent model system for probing how the heme environment tunes NO dynamics and for comparing it to the endothelial NO synthase heme domain (eNOS(HD)) using ultrafast transient spectroscopy. NO rebinding in HNS from Staphylococcus aureus (SA-HNS) is faster than that measured for either Bacillus anthracis (BA-HNS) or for eNOS(HD) in both oxidized and reduced forms in the presence of arginine. To test whether these distinct rates arise from different energy barriers for NO recombination, we measured rebinding kinetics at several temperatures. Our data are consistent with different barriers for NO recombination in SA-HNS and BA-HNS and the presence of a second NO-binding site. The hypothesis that an additional NO-binding cavity is present in BA-HNS is also consistent with the effect of the NO concentration on its rebinding. The lack of the effect of NO concentration on the geminate rebinding in SA-HNS could be due to an isolated second site. We confirm the existence of a second NO site in the oxygenase domain of the reduced eNOS as previously hypothesized [A. Slama-Schwok, M. Négrerie, V. Berka, J.C. Lambry, A.L. Tsai, M.H. Vos, J.L. Martin, Nitric oxide (NO) traffic in endothelial NO synthase. Evidence for a new NO binding site dependent on tetrahydrobiopterin? J. Biol. Chem. 277 (2002) 7581-7586]. This site requires the presence of arginine and BH(4); and we propose that NO dynamic and escape from eNOS is regulated by the active site H-bonding network connecting between the heme, the substrate, and cofactor.
一些革兰氏阳性细菌病原体含有一个基因，该基因编码一种蛋白质（HNS，一氧化氮合酶样蛋白的血红素结构域），其与哺乳动物一氧化氮合酶（NOS）的加氧酶结构域具有显著的序列同一性。然而，它们缺乏参与哺乳动物同工型中活性二聚体稳定性的N末端和锌 - 半胱氨酸基序。HNS的独特性质使其成为一个出色的模型系统，用于探究血红素环境如何调节一氧化氮动力学，并使用超快瞬态光谱将其与内皮型一氧化氮合酶血红素结构域（eNOS(HD)）进行比较。在金黄色葡萄球菌（SA - HNS）中，一氧化氮重新结合的速度比炭疽芽孢杆菌（BA - HNS）或在存在精氨酸的情况下氧化和还原形式的eNOS(HD)所测得的速度都要快。为了测试这些不同的速率是否源于一氧化氮重组的不同能量障碍，我们在几个温度下测量了重新结合动力学。我们的数据与SA - HNS和BA - HNS中一氧化氮重组的不同障碍以及第二个一氧化氮结合位点的存在相一致。BA - HNS中存在额外一氧化氮结合腔的假设也与一氧化氮浓度对其重新结合的影响相一致。一氧化氮浓度对SA - HNS中双生重新结合缺乏影响可能是由于一个孤立的第二个位点。我们证实了如先前假设的那样，在还原型eNOS的加氧酶结构域中存在第二个一氧化氮位点[A. Slama - Schwok, M. Négrerie, V. Berka, J.C. Lambry, A.L. Tsai, M.H. Vos, J.L. Martin, 内皮型一氧化氮合酶中的一氧化氮（NO）运输。是否存在一个依赖于四氢生物蝶呤的新的NO结合位点的证据？《生物化学杂志》277 (2002) 7581 - 7586]。这个位点需要精氨酸和BH(4)的存在；并且我们提出一氧化氮的动力学和从eNOS的逸出是由连接血红素、底物和辅因子的活性位点氢键网络调节的。
