SPECTRAL AND KINETIC-STUDIES ON THE ACTIVATION OF SOLUBLE GUANYLATE-CYCLASE BY NITRIC-OXIDE

The soluble form of guanylate cyclase (sGC) is the only definitive rec eptor for the signaling agent nitric oxide (. NO). The enzyme is a het erodimer of homologous subunits in which each subunit binds 1 equiv of 5-coordinate high-spin heme. . NO increases the V-max of sGC up to 40 0-fold and has previously been shown to bind to the heme to form a 5-c oordinate complex. Using stopped-flow spectrophotometry, it is demonst rated that the binding of . NO to the heme of sGC is a complex process . . NO first binds to the heme to form a 6-coordinate nitrosyl complex , which then converts to a 5-coordinate nitrosyl complex through one o f two ways. For 28 +/- 4% of the heme, the 6-coordinate nitrosyl compl ex rapidly (similar to 20 s(-1)) converts to the 5-coordinate complex. For the remaining 72 +/- 4% of the heme, the conversion of the 6-coor dinate nitrosyl complex to a 5-coordinate nitrosyl complex is slow (0. 1-1.0 s(-1)) and is dependent upon the interaction of NO with an unide ntified non-heme site on the protein. The heme (200 nM) was completely converted to the 5-coordinate state with as little as 500 nM . NO, an d the equilibrium dissociation constant of NO for activating the enzym e was determined to be less than or equal to 250 nM. Gel-filtration an alysis indicates that the binding of . NO to the heme has no effect on the native molecular mass of the protein. Correlation of electronic a bsorption spectra with activity measurements indicates that the 5-coor dinate nitrosyl form of the enzyme is activated relative to the restin g 5-coordinate ferrous form of the enzyme.