Tonic and acute nitric oxide signaling through soluble guanylate cyclase is mediated by nonheme nitric oxide, ATP, and GTP

doi:10.1073/pnas.0506289102

Tonic and acute nitric oxide signaling through soluble guanylate cyclase is mediated by nonheme nitric oxide, ATP, and GTP

Departments of ^†Biological Chemistry and ^§Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109; ^¶Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720; and ^∥Division of Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Communicated by Jack E. Dixon, University of California at San Diego, La Jolla, CA, July 22, 2005 (received for review May 27, 2005)

Abstract

Nitric oxide (NO) affects many physiological systems by activating cGMP signaling cascades through soluble guanylate cyclase (sGC). In the accepted model, NO binds to the sGC heme, activating the enzyme. Here, we report that in the presence of physiological concentrations of ATP and GTP, NO dissociation from the sGC heme is ≈160 times slower than the rate of enzyme deactivation in vitro. Deactivated sGC still has NO bound to the heme, and full activation requires additional NO. We propose an activation model where, in the presence of both ATP and GTP, tonic NO forms a stable heme complex with low sGC activity; acute production of NO transiently and fully activates this NO-bound sGC.

Footnotes

↵ †† To whom correspondence should be addressed at: Department of Chemistry, University of California, 211 Lewis Hall, Berkeley, CA 94720-1460. E-mail: marletta{at}berkeley.edu.
↵ ‡ S.P.L.C. and J.A.W. contributed equally to this work.
Author contributions: S.P.L.C., J.A.W., and M.A.M. designed research; S.P.L.C. and J.A.W. performed research; S.P.L.C., J.A.W., and M.A.M. analyzed data; and S.P.L.C., J.A.W., and M.A.M. wrote the paper.
Abbreviations: β2(1–217), residues 1–217 of the soluble guanylate cyclase β2 isoform; DEA/NO, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; EIA, enzyme immunoassay; GMPCPP, guanosine 5′-[α,β-methylene]triphosphate; NO, nitric oxide; PROLI/NO, disodium 1-(hydroxy-NNO-azoxy)-l-proline; sGC, soluble guanylate cyclase.
↵ ‡‡ For activity studies, we used the GTP analogue GMPCPP, which is not turned over by sGC, for premixing instead of GTP. Premixing with low concentrations of GMPCPP produces the same effect as GTP in spectral studies and does not interfere with enzyme activity.