ACTIVATION MECHANISM OF SOLUBLE GUANILATE CYCLASE FROM BOVINE LUNG

从牛肺中纯化出可溶性鸟苷酸环化酶，具有明显的同质性。纯化后的酶为异源二聚体，含有1个原血红素IX/异源二聚体。酶血红素的光谱分析表明，铁、亚铁和亚铁NO形态为5配位态，而CO和氰化物形态为6配位态。EPR研究证实，铁酶处于纯5位高自旋态，与氰化物结合后转化为6位低自旋态。NO配合物表现出典型的5坐标状态的3线EPR信号。在这些被检测的物种中，亚铁NO配合物仅表现出明显的活性，而其他物种除了CO配合物的活性比基态高5倍外，几乎没有活性。当用停止流动法检测NO与铁酶的结合时，发现瞬时形成一个Soret峰为419 nm的6位NO配合物，然后转化为半衰期约为25 msec的5位NO配合物。NO与铁酶的结合速率常数大于10^7 M^<-1> sec^<-1>，是CO的结合速率常数的1000倍。这些结果表明，血红素结合的NO触发铁-近端配体键的减弱或断裂，导致5位NO复合物的形成。因此，通过NO结合对铁近端配体键的调节是激活的必要条件，但包括CO在内的其他配体的结合并没有引起铁近端配体键的明显变化。

Soluble guanylate cyclase has been purified to apparent homogeneity from bovine lung. The purified enzyme was a heterodimer and contained 1 protoheme IX/heterodimer. Optical spectral analyzes of the enzyme heme suggested that the ferric, ferrous and ferrous NO forms were 5-coordinate, while ferrous CO and cyanide forms were in a 6-coordinate state. EPR studies confirmed that the ferric enzyme was in a pure 5-coordinate high spin state, which converted to a 6-coordinate low spin state upon binding of cyanide. The NO complex exhibited the 3-line EPR signal typical of a 5-coordinate state. Among these species examined, the ferrous NO complex only exhibited a marked activity, while other species were practically inactive except for the CO complex being 5 times more active than the basal state.When the binding of NO to the ferrous enzyme was examined by a stopped flow method, a 6-coordinate NO complex with 419 nm Soret peak was found to be transiently formed and then converted to the 5-coordinate NO complex with a half life of about 25 msec. The binding rate constant of NO to the ferrous enzyme was estimated over 10^7 M^<-1> sec^<-1>, which was about 1000 times greater than that for the CO binding. These results indicate that the heme bound NO triggers the weakening or breaking of the iron-proximal ligand bond, resulting in the formation of the 5-coordinate NO complex. Thus, the modulation of the iron-proximal ligand bond by the NO binding was essential for the activation, but the binding of other ligands including CO did not cause appreciable changes in the iron-proximal bond.