Jr. Stone et Ma. Marletta, SPECTRAL AND KINETIC-STUDIES ON THE ACTIVATION OF SOLUBLE GUANYLATE-CYCLASE BY NITRIC-OXIDE, Biochemistry, 35(4), 1996, pp. 1093-1099
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.