Weisslocker-Schaetzel Marine, Lembrouk Mehdi, Santolini Jérôme, Dorlet Pierre
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay , F-91198 Gif-sur-Yvette cedex, France.
Biochemistry. 2017 Feb 7;56(5):748-756. doi: 10.1021/acs.biochem.6b01018. Epub 2017 Jan 20.
Nitric oxide is produced in mammals by the nitric oxide synthase (NOS) isoforms at a catalytic site comprising a heme associated with a biopterin cofactor. Through genome sequencing, proteins that are highly homologous to the oxygenase domain of NOSs have been identified, in particular in bacteria. The active site is highly conserved except for a valine residue in the distal pocket that is replaced with an isoleucine in bacteria. This switch was previously reported to influence the kinetics of the reaction. We have used the V346I mutant of the mouse inducible NOS (iNOS) as well as the I224V mutant of the NOS from Bacillus subtilis (bsNOS) to study their spectroscopic signatures in solution and look for potential structural differences compared to their respective wild types. Both mutants seem destabilized in the absence of substrate and cofactor. When both substrate and cofactor are present, small differences can be detected with N-hydroxy-l-arginine compared to arginine, which is likely due to the differences in the hydrogen bonding network of the distal pocket. Stopped-flow experiments evidence significant changes in the kinetics of the reaction due to the mutation as was already known. We found these effects particularly marked for iNOS. On the basis of these results, we performed rapid freeze-quench experiments to trap the biopterin radical and found the same results that we had obtained for the wild types. Despite differences in kinetics, a radical could be trapped in both steps for the iNOS mutant but only for the first step in the mutant of bsNOS. This strengthens the hypothesis that mammalian and bacterial NOSs may have a different mechanism during the second catalytic step.
在哺乳动物中，一氧化氮由一氧化氮合酶（NOS）同工型在一个催化位点产生，该催化位点包含一个与生物蝶呤辅因子相关的血红素。通过基因组测序，已鉴定出与NOS加氧酶结构域高度同源的蛋白质，尤其是在细菌中。除了远端口袋中的一个缬氨酸残基在细菌中被异亮氨酸取代外，活性位点高度保守。此前报道这种转变会影响反应动力学。我们使用了小鼠诱导型NOS（iNOS）的V346I突变体以及枯草芽孢杆菌NOS（bsNOS）的I224V突变体来研究它们在溶液中的光谱特征，并寻找与各自野生型相比潜在的结构差异。在没有底物和辅因子的情况下，两种突变体似乎都不稳定。当同时存在底物和辅因子时，与精氨酸相比，用N-羟基-L-精氨酸可以检测到微小差异，这可能是由于远端口袋氢键网络的差异所致。停流实验证明，正如已知的那样，突变会导致反应动力学发生显著变化。我们发现这些效应在iNOS中尤为明显。基于这些结果，我们进行了快速冷冻淬灭实验以捕获生物蝶呤自由基，得到了与野生型相同的结果。尽管动力学存在差异，但iNOS突变体在两个步骤中都能捕获自由基，而bsNOS突变体仅在第一步能捕获自由基。这强化了这样一种假设，即哺乳动物和细菌的NOS在第二个催化步骤中可能具有不同的机制。
