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 receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer of homologous subunits in which each subunit binds 1 equiv of 5-coordinate high-spin heme. .NO increases the Vmax of sGC up to 400-fold and has previously been shown to bind to the heme to form a 5-coordinate complex. Using stopped-flow spectrophotometry, it is demonstrated 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 of two ways. For 28 +/- 4% of the heme, the 6-coordinate nitrosyl complex rapidly (approximately 20 s-1) converts to the 5-coordinate complex. For the remaining 72 +/- 4% of the heme, the conversion of the 6-coordinate 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 unidentified 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, and the equilibrium dissociation constant of .NO for activating the enzyme was determined to be < or = 250 nM. Gel-filtration analysis indicates that the binding of .NO to the heme has no effect on the native molecular mass of the protein. Correlation of electronic absorption spectra with activity measurements indicates that the 5-coordinate nitrosyl form of the enzyme is activated relative to the resting 5-coordinate ferrous form of the enzyme.
Stone
J R
JR
Department of Biological Chemistry, School of Medicine, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Marletta
M A
MA
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006152:Cyclic GMP / Q000032:analysis; D004789:Enzyme Activation; D017022:Flow Injection Analysis; D006162:Guanylate Cyclase / Q000187:drug effects / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008970:Molecular Weight; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction; D012995:Solubility; D013053:Spectrophotometry
D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase; D006152:Cyclic GMP