STRUCTURAL-CHANGES IN THE HEME PROXIMAL POCKET INDUCED BY NITRIC-OXIDE BINDING TO SOLUBLE GUANYLATE-CYCLASE

When expressed in Escherichia coli, the heme domain [beta 1(1-385)] of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiom etric amount of bound heme [Zhao, Y., and Marletta, M. A. (1997) Bioch emistry 36, 15959-15964]. Nitric oxide (NO) binding to the heme in bet a 1(1-385) leads to cleavage of the Fe-His bond and formation of a fiv e-coordinate NO-heme complex. Addition of imidazole to the five-coordi nate NO complex shifts the Soret peak from 399 to 420 nm, which appear s 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 putativ e six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coor dinate NO complex with two nitrogen atoms as the axial ligands. [N-15] 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 imi dazole molecule. These results show that NO binding to sGC not only le ads to the cleavage of the Fe-His bond but also induces a conformation al change which opens the heme proximal pocket large enough to accommo date an exogenous imidazole molecule. These observations have importan t implications for determining the NO activation mechanism of sGC.