Expression and Characterization of the Catalytic Domains of Soluble Guanylate Cyclase: Interaction with the Heme Domain ² Jonathan A. Winger ‡ and Michael A. Marletta* ,‡,§ Department of Medicinal Chemistry, The UniVersity of Michigan, Ann Arbor, Michigan 48109-0606, and Department of Chemistry, Department of Molecular and Cell Biology, DiVision of Physical Biosciences, Lawrence Berkeley National Laboratory, UniVersity of California, Berkeley 94720-1460 ReceiVed NoVember 12, 2004; ReVised Manuscript ReceiVed December 16, 2004 ABSTRACT: The catalytic domains (R cat and  cat ) of R11 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. R cat ,  cat , and the R cat  cat heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. R cat and  cat were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when R cat and  cat were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full- length enzyme, the R cat  cat complex was more active in the presence of Mn 2+ as compared to the physiological cofactor Mg 2+ , although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K M for Mn 2+ -GTP was measured to be 85 ( 18 µM, and in the presence of Mn 2+ -GTP, the K D for the R cat  cat complex was 450 ( 70 nM. The N-terminal heme- bound regulatory domain of the 1 subunit of sGC inhibited the activity of the R cat  cat complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed. The principle physiological receptor for nitric oxide (NO) when functioning as a signaling agent is soluble guanylate cyclase (sGC, 1 EC 4.6.1.2) (1-5). sGC catalyzes a cycliza- tion reaction in which guanosine 5′-triphosphate (GTP) is converted into the 3′,5′-cyclic monophosphate second mes- senger, cGMP. The product cGMP elicits downstream effects that ultimately result in phenomena such as smooth muscle relaxation and vasodilation (6). An understanding of the molecular details of mechanism and regulation of sGC is of interest in developing therapeutic strategies for the treatment of NO/cGMP signaling pathway-related disease. sGC is a heterodimeric heme-containing protein consisting of R and  subunits. Each subunit contains a conserved N-terminal regulatory domain (7) predicted to exhibit a novel structure (8), the H-NOX (heme-nitric oxide/oxygen bind- ing) fold. It is this domain to which the heme is bound in the  subunit of the sGC R11 heterodimer. Upon binding of NO to the heme, the enzyme is activated several hundred fold above the basal level (9, 10). The changes induced in the sGC heme environment by the binding of NO are thought to be responsible for initiating the events that result in enzyme activation; however, the exact mechanism of activa- tion remains unclear. The corresponding N-terminal region of the R1 subunit is homologous to the N-terminus of the 1 subunit and likely represents a similar fold despite the inability to bind heme. sGC truncations consisting solely of the 1H-NOX domain appear monomeric (D. S. Karow, J. H. Davis, and M. A. Marletta, manuscript in preparation), and the corresponding domain of the R1 subunit can be deleted from the full-length enzyme with little effect on enzyme activity (11), indicating that the H-NOX domains are not likely to be involved in dimerization. Each subunit also contains a central region of unknown function consisting of a domain predicted to adopt a PAS fold and an amphi- pathic helical domain (7). Recent evidence suggests that these central domains are involved in sensitivity to NO and the exogenous activator, YC-1 (11), and in hetero- and homo- ² Funding was provided by the LDRD Fund from LBNL to M.A.M. and by an Eli Lilly Fellowship, a Pharmaceutical Sciences Training Program Fellowship (grant number GM07767 NIGMS), and an American Foundation for Pharmaceutical Education Fellowship to J.A.W. * To whom correspondence should be addressed at University of California, Berkeley, Department of Chemistry, 211 Lewis Hall, Berkeley, CA 94720-1460. Phone: (510) 643-9325; fax: (510) 643- 9388; e-mail: marletta@berkeley.edu. ‡ University of Michigan. § University of California. 1 Abbreviations: sGC, soluble guanylate cyclase; NO, nitric oxide; GTP, guanosine 5′-triphosphate; cGMP, guanosine 3′,5′-cyclic mono- phosphate; PAS, protein fold first identified in the Per, Arnt, and Sim proteins; AC, adenylate cyclase; R cat, the catalytic domain of the sGC R1 subunit; cat, the catalytic domain of the sGC 1 subunit; E. coli, Escherichia coli; IPTG, isopropyl--D-thiogalactopyranoside; DEA/ NO, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-di- olate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis; DEA, diethanolamine; DTT, dithiothreitol; HEPES, 4-(2- hydroxyethyl)-1-piperazineethane sulfonic acid; PVDF, polyvinylidene fluoride; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol; PCR, poly- merase chain reaction; EIA, enzyme immunoassay. 4083 Biochemistry 2005, 44, 4083-4090 10.1021/bi047601d CCC: $30.25 © 2005 American Chemical Society Published on Web 02/17/2005