Spectral and ligand-binding properties of an unusual hemoprotein, the ferric form of soluble guanylate cyclase.
The soluble form of guanylate cyclase (sGC) is a hemoprotein which serves as the only known receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin type b heme. .NO increases the Vmax 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 displays 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 electronic absorption spectroscopy, it was observed that the ferric heme binds cyanide to form a 6-coordinate low-spin complex. The rate constants for association (k(on)) and dissociation (k(off)) of cyanide at 10 degrees 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 of the enzyme, which has a low affinity for ligands that form 6-coordinate complexes due to an unusually fast off-rate, the ferric form of the enzyme appears to have a low affinity for ligands due to a slow on-rate. The ferric heme binds azide with a Kd of 26 +/- 4 mM to form a high-spin complex. The ferric form of the enzyme has a specific activity of approximately 57% that of the nonactivated ferrous form of the enzyme. However, in contrast to the mild activation of the ferrous enzyme by carbon monoxide, the ferric enzyme is not activated by cyanide. These results indicate that there may be a significant structural change in the protein upon the oxidation of the heme iron.
Stone
J R
JR
Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor 48109-1065, USA.
Sands
R H
RH
Dunham
W R
WR
Marletta
M A
MA
D001386:Azides / Q000378:metabolism; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011190:Potassium Cyanide / Q000378:metabolism; D019810:Sodium Azide; D012995:Solubility; D013053:Spectrophotometry
D001386:Azides; D005290:Ferric Compounds; D006420:Hemeproteins; D008024:Ligands; D019810:Sodium Azide; D006162:Guanylate Cyclase; D011190:Potassium Cyanide