Interaction of home oxygenase-2 with nitric oxide donors - Is the oxygenase an intracellular 'sink' for NO?
Interaction of home oxygenase-2 with nitric oxide donors - Is the oxygenase an intracellular 'sink' for NO?
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DOI：
10.1046/j.1432-1327.1999.00677.x
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发表时间：
1999-09-01
期刊：
影响因子：
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通讯作者：
Maines, MD
中科院分区：
文献类型：
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作者：
Ding, Y;McCoubrey, WK;Maines, MD
血红素加氧酶-2(HO-2)是热休克蛋白-32家族的组成同源蛋白。这些蛋白质催化血红素氧化裂解成一氧化碳和胆绿素，并释放铁。HO-2是一种血红素蛋白，它与血红素调节基序(HRMS)上的血红素与保守的Cys-Pro对结合；HO-2中存在两个HRM副本(Cys264和Cys281)。HO-2 HRMS不存在于HO-1中，也不参与HO-2的催化活性。用光学CD、光谱分析和活性分析检测了HO同工酶与NO供体产生的NO物种的反应活性。使用纯化的大肠杆菌表达的HO制剂、野生型HO-2、Cys264/Cys281-->；Ala/Ala HO-2突变体(HO-2-mut)和I-IO-I制剂。野生型HO-2细胞经硝普钠、S-亚硝基-谷胱甘肽、S-亚硝基-N-乙酰青霉胺或3-吗啉磺基-农亚胺处理后，Soret带发生了II型红移，而NO清除剂羟钴胺(HcB)则阻止了这种红移。只有SIN-I通过同时产生NO和超氧阴离子来产生过氧亚硝酸根，降低了HO-2的Soret区吸收和吡啶血色素原光谱；超氧化物歧化酶(SOD)阻止了这种下降。血红素与HO-2蛋白的结合需要转移和/或减少SIGER条带的吸收。NO供体显著抑制HO-2活性，SNP是最有效的抑制因子(>；40%)。再一次，用HCB捕获NO阻止了HO-2的失活。HO-1和HO-2-mut未被任何供体灭活。CD数据表明，HO-2活性的降低与HO-2二级结构中没有物种的变化有关。Western印迹分析表明，NO供体没有导致IIO-L蛋白丢失，SIN-I和SNP处理的HeLa细胞的Northern印迹分析表明，与对处理有显著反应的HO-1mRNA不同，NO供体HO-2mRNA水平略有增加(约2-3倍)。这些数据与NO与HO-2结合的血红素相互作用影响底物结合和/或氧活化涉及的残基的电子相互作用的可能性是一致的。这些发现支持这样的假设，即HO-2和NO是反式抑制物，因此NO的生物活性通过与HO-2的相互作用而减弱，从而成为血红素配体的细胞内“汇”，而NO抑制HO-2的催化活性。因此，CO和NO这两种信号分子的细胞水平将受到抑制。
Heme oxygenase-2(HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2 mutant (HO-2-mut) and I-IO-I preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-I); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-I, which produces peroxynitrite by generating both No and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Sorer band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause IIO-l protein loss and Northern blot analysis of HeLa cells treated with SIN-I and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased (approximate to two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules,CO and NO would be moderated.