Structural Changes in the Heme Proximal Pocket Induced by Nitric Oxide Binding to Soluble Guanylate Cyclase † Yunde Zhao, ‡ Curtis Hoganson, § Gerald T. Babcock,* ,§ and Michael A. Marletta* ,‡,|,⊥ Department of Biological Chemistry, School of Medicine, Howard Hughes Medical Institute, and Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, The UniVersity of Michigan, Ann Arbor, Michigan 48109-1065, and Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824-1322 ReceiVed May 18, 1998; ReVised Manuscript ReceiVed July 8, 1998 ABSTRACT: When expressed in Escherichia coli, the heme domain [1(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 1(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. [ 15 N]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. Soluble guanylate cyclase (sGC) 1 plays an essential role in nitric oxide (NO) signaling by functioning as a NO receptor. NO has been shown to be involved in many biological processes, including neuronal signaling, vasodi- latation, and the host response to infection (2, 3). sGC catalyzes the generation of the second messenger cGMP from GTP (for reviews, see (refs 4 and 5). cGMP has been shown to regulate various cellular signal transduction pathways, including activation of cGMP-dependent protein kinases (6). In the presence of NO, sGC is activated up to 400-fold (7). The molecular details of both activation and deactivation of sGC are not fully understood. sGC is a heterodimeric hemoprotein composed of R1 and 1 subunits when it is isolated from lung tissue (8-12). The heme binding region and the catalytic site are localized in different regions of the protein. The heme binding pocket is formed from residues in the N-terminal region of the 1 subunit (1), while the catalytic site(s) is located in the C-terminal region of the R1 and 1 subunits (13). Coexpression of the C-terminal fragments of R1 and 1[R1(367-691) and 1(306-619)] in COS cells was found to be sufficient for generating basal sGC activity, but not the NO-stimulated activity (13). We have previously demonstrated that the N-terminal fragment of the 1 subunit [1(1-385)] when it is expressed in Escherichia coli, is isolated with 1 equiv of bound heme (1). The electronic absorption (1) and resonance Raman spectra (14) of 1(1-385) are almost identical to those of heterodimeric sGC, indicating that the catalytic domains of the 1 subunit and the R1 subunit are not required for the formation of the heme binding pocket. Therefore, 1(1- 385) was used in this study to characterize the interaction between NO and the heme of sGC. When isolated from lung tissue (12, 15, 16) or recombinant baculovirus and/or 9 cells (P. E. Brandish and M. A. Marletta, unpublished results), sGC contains a ferrous high-spin, five-coordinate heme with a histidine residue as the only axial ligand as indicated by both electronic absorption and resonance Raman spectro- scopic studies. We have further identified histidine 105 (H105) in the 1 subunit as the heme proximal ligand using site-directed mutagenesis and spectroscopic methods (14). Activation of sGC by NO is mediated by the interaction of NO and the heme. Heme-deficient sGC retains basal sGC activity, but has lost its ability to respond to NO (17). NO readily forms a nitrosyl complex with the heme of sGC and shifts the Soret maximum from 431 to 399 nm (12). EPR (18), electronic absorption (12), and resonance Raman spectroscopic studies (15, 16, 19, 20) have shown that the sGC-NO complex is five-coordinate with NO as the only † The studies were supported by the Howard Hughes Medical Institute, the Searle chair endowment fund, and NIH Grant GM25480. * To whom correspondence should be addressed. ‡ Department of Biological Chemistry, The University of Michigan. § Michigan State University. | Howard Hughes Medical Institute, The University of Michigan. ⊥ Interdepartmental Program in Medicinal Chemistry, The University of Michigan. 1 Abbreviations: cGMP, guanosine 3′,5′-cyclic monophosphate; CO, carbon monoxide; deoxyMb, ferrous myoglobin; DTT, dithiothreitol; EPR, electron paramagnetic resonance; GTP, guanosine 5′-triphosphate; Im, imidazole; 2-MeIm, 2-methylimidazole; 4-MeIm, 4-methylimida- zole; N-MeIm, N-methylimidazole; NO, nitric oxide; sGC, soluble guanylate cyclase. 12458 Biochemistry 1998, 37, 12458-12464 S0006-2960(98)01156-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/19/1998