Structural changes in the heme proximal pocket induced by nitric oxide binding to soluble guanylate cyclase.
When expressed in Escherichia coli, the heme domain [beta1(1-385)] of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiometric amount of bound heme [Zhao, Y., and Marletta, M. A. (1997) Biochemistry 36, 15959-15964]. Nitric oxide (NO) binding to the heme in beta1(1-385) leads to cleavage of the Fe-His bond and formation of a five-coordinate NO-heme complex. Addition of imidazole to the five-coordinate NO complex shifts the Soret peak from 399 to 420 nm, which appears to result from the formation of a six-coordinate NO complex. Removal of the added imidazole by gel filtration results in formation of the five-coordinate NO complex once again. The EPR spectrum of the putative six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coordinate NO complex with two nitrogen atoms as the axial ligands. [15N]Imidazole simplifies the six-coordinate NO complex EPR spectrum to six distinct derivative-shaped lines (a triplet of doublets), indicating that the other axial ligand in the six-coordinate NO complex is an imidazole molecule. These results show that NO binding to sGC not only leads to the cleavage of the Fe-His bond but also induces a conformational change which opens the heme proximal pocket large enough to accommodate an exogenous imidazole molecule. These observations have important implications for determining the NO activation mechanism of sGC.
Zhao
Y
Y
Department of Biological Chemistry, School of Medicine, Howard Hughes Medical Institute, The University of Michigan, Ann Arbor 48109-1065, USA.
Hoganson
C
C
Babcock
G T
GT
Marletta
M A
MA
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006162:Guanylate Cyclase / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D046911:Macromolecular Substances; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012995:Solubility
D007093:Imidazoles; D046911:Macromolecular Substances; D010446:Peptide Fragments; D009569:Nitric Oxide; D006418:Heme; C029899:imidazole; D006162:Guanylate Cyclase; C018100:1-methylimidazole; C023439:4-methylimidazole