title: A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase.
abstract: Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV-vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k                         (1) but a larger k                         (2). Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.
authors: Department of Chemistry, Fudan University, Shanghai, China.
authors: Fangfang
authors: F
authors: Zhong
authors: Jie
authors: J
authors: Pan
authors: Xiaoxiao
authors: X
authors: Liu
authors: Hongyan
authors: H
authors: Wang
authors: Tianlei
authors: T
authors: Ying
authors: Jihu
authors: J
authors: Su
authors: Zhong-Xian
authors: ZX
authors: Huang
authors: Xiangshi
authors: X
authors: Tan
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics*; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
chemical_list: D006420:Hemeproteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase