Lin Hsia-Lien, Myshkin Eugene, Waskell Lucy, Hollenberg Paul F
Department of Pharmacology, University of Michigan and VA Medical Center, Ann Arbor, Michigan 48109, USA .
Chem Res Toxicol. 2007 Nov;20(11):1612-22. doi: 10.1021/tx700220e. Epub 2007 Oct 2.
This study examined the reaction of peroxynitrite (PN) with two human cytochrome P450s, P450 2B6 (2B6) and P450 2E1 (2E1). After the reaction with PN, the NADPH/reductase-supported 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) deethylation activity of both P450s was decreased in a concentration-dependent manner. HPLC analysis revealed that the prosthetic heme group of 2B6 was modified but to a lesser extent than the decrease in enzymatic activity. In contrast, the heme moiety of 2E1 was not altered. These results suggest that protein modification by PN contributed to the loss in enzymatic activity of 2B6 and 2E1 but to different extents. After trypsin digestion of the control and PN-inactivated P450s, tyrosine nitration was used as a biomarker for protein modification and the addition of the nitro group was determined using electrospray ionization-liquid chromatography-tandem mass spectrometry, allowing site-specific assignment of the tyrosine residues nitrated. Tyrosine residues 354, 244, 268, and 380 in 2B6 and tyrosine residues 317, 422, 69, and 380 in 2E1 were found to be nitrated. Tyrosine 354 is the primary site of nitration in 2B6, and tyrosine residues 422 and 317 are the primary targets for nitration in 2E1. After PN exposure, the EFC catalytic activity of 2E1 supported by tert-butylhydroperoxide was not affected, and the activity of 2B6 supported by tert-butylhydroperoxide was decreased to a lesser extent than that supported by NADPH/reductase. Following exposure to PN, the levels of the reduced-CO complex were less than the content of native heme remaining. These results suggest that PN-mediated protein modification has no effect on substrate binding but may impair the interaction of the reductase with P450s, thereby inhibiting electron transfer. Homology modeling shows that Tyr422 of 2E1 is in close proximity to the FMN domain of reductase, suggesting that Tyr422 may be involved in transferring electrons from the reductase to the heme and thus may play a critical structural and functional role in the extensive activity loss following PN exposure.
本研究检测了过氧亚硝酸盐（PN）与两种人细胞色素P450，即P450 2B6（2B6）和P450 2E1（2E1）的反应。与PN反应后，两种P450由NADPH/还原酶支持的7-乙氧基-4-（三氟甲基）香豆素（EFC）脱乙基活性均呈浓度依赖性降低。高效液相色谱分析显示，2B6的辅基血红素基团发生了修饰，但程度小于酶活性的降低。相比之下，2E1的血红素部分未发生改变。这些结果表明，PN对蛋白质的修饰导致了2B6和2E1酶活性的丧失，但程度不同。在对对照和PN失活的P450进行胰蛋白酶消化后，酪氨酸硝化被用作蛋白质修饰的生物标志物，并使用电喷雾电离-液相色谱-串联质谱法测定硝基的添加情况，从而实现对硝化的酪氨酸残基进行位点特异性定位。发现2B6中的酪氨酸残基354、244、268和380以及2E1中的酪氨酸残基317,、422、69和380被硝化。酪氨酸354是2B6中硝化的主要位点，酪氨酸残基422和317是2E1中硝化的主要靶点。PN暴露后，叔丁基过氧化氢支持的2E1的EFC催化活性未受影响，叔丁基过氧化氢支持的2B6的活性降低程度小于NADPH/还原酶支持的情况。暴露于PN后，还原型一氧化碳复合物的水平低于剩余天然血红素的含量。这些结果表明，PN介导的蛋白质修饰对底物结合没有影响，但可能损害还原酶与P450的相互作用，从而抑制电子传递。同源性建模显示，2E1的Tyr422靠近还原酶的FMN结构域，表明Tyr422可能参与将电子从还原酶转移至血红素，因此可能在PN暴露后广泛的活性丧失中发挥关键的结构和功能作用。
