| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
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.
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