Jr. Stone et al., SPECTRAL AND LIGAND-BINDING PROPERTIES OF AN UNUSUAL HEMOPROTEIN, THEFERRIC FORM OF SOLUBLE GUANYLATE-CYCLASE, Biochemistry, 35(10), 1996, pp. 3258-3262
The soluble form of guanylate cyclase (sGC) is a hemoprotein which ser
ves as the only known receptor for the signaling agent nitric oxide ((
NO)-N-.), The enzyme is a heterodimer in which each subunit binds 1 eq
uiv of 5-coordinate high-spin type b heme. (NO)-N-. increases the V-ma
x of sGC up to 400-fold by binding to the heme to form a 5-coordinate
ferrous nitrosyl complex. The electron paramagnetic resonance spectrum
of the ferric form of the enzyme has been obtained. The spectrum disp
lays rhombic symmetry and is indicative of a high-spin heme. Computer
simulation of the EPR spectrum yields g values of 6.36, 5.16, and 2.0
with linewidths of 3.3, 4.1, and 3.3 mT, respectively, Using electroni
c absorption spectroscopy, it was observed that the ferric heme binds
cyanide to farm a 6-coordinate low-spin complex. The rate constants fo
r association (k(on)) and dissociation (k(off)) of cyanide at 10 degre
es C have been determined to be (7.8 +/- 0.3) x 10(-2) M(-1) s(-1) and
(7.2 +/- 0.2) x 10(-5) s(-1). respectively. Unlike the ferrous form o
f the enzyme, which has a low affinity for ligands that form 6-coordin
ate complexes due to an unusually fast off-rate, the ferric form of th
e enzyme appears to have a low affinity for ligands due to a slow on-r
ate. The ferric heme binds azide with a K-d of 26 +/- 4 mM to form a h
igh-spin complex. The ferric form of the enzyme has a specific activit
y of similar to 57% that of the nonactivated ferrous farm of the enzym
e, However, in contrast to the mild activation of the ferrous enzyme b
y carbon monoxide, the ferric enzyme is not activated by cyanide. Thes
e results indicate that there may be a significant structural change i
n the protein upon the oxidation of the heme iron.