CHARACTERIZATION OF 1H-[1,2,4]OXADIAZOLO[4,3-A]QUINOXALIN-1-ONE AS A HEME-SITE INHIBITOR OF NITRIC OXIDE-SENSITIVE GUANYLYL CYCLASE

Nitric oxide (NO) binds with high affinity to the heme of soluble guan ylyl cyclase (sGC), resulting in accumulation of the second messenger cGMP in many biological systems. 1H-[1,2,4]Ox-adiazolo[4,3-a]quinoxali n-1-one (ODQ) was recently described as potent and selective inhibitor of sGC, providing an invaluable tool with which to settle the functio n of the cGMP pathway in NO-mediated signal transduction [Mol. Pharmac ol. 48:184-188 (1995)]. The present study investigated the mechanism o f ODQ-induced inhibition of purified bovine lung sGC. The drug induced a rightward shift of the concentration-response curves recorded with two different NO donors and a reduction of maximal sGC activity, point ing to a mixed type of inhibition. The time course of NO-stimulated sG C activity determined in the presence of 0.3 mu M ODQ showed that the inhibitory effect was time-dependent (half-time similar to 3 min) and virtually complete after about 10 min. The cyclase did nor recover fro m ODQ-induced inhibition upon extensive dilution, pointing to an appar ently irreversible inactivation of the enzyme by the quinoxalin. Light absorbance spectroscopy showed that ODQ (0.3 mM) induced a shift of t he Sorer band of the heme from 431 nm to 393 nm, indicating that ODQ o xidizes the ferrous form of the enzyme to the ferric species, which is thought to exhibit only poor NO sensitivity. Together, our results su ggest that inhibition of sGC by ODQ is NO-competitive and results in a n apparently irreversible oxidation of the prosthetic heme group.