科研之友
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摘要
Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an alpha beta 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 alpha and beta subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC alpha 1 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 alpha 1 subunit and the beta 1 subunit were investigated via heme reconstitution, UV-vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the alpha 1 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 sGC beta 195 by simple one-step binding, whereas CO binds to human sGC alpha 259, 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 sGC alpha 259 has a smaller k (1) but a larger k (2). Additionally, the role of the cavity of the alpha 1 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.
