A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase |
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Authors: | Fangfang Zhong Jie Pan Xiaoxiao Liu Hongyan Wang Tianlei Ying Jihu Su Zhong-Xian Huang Xiangshi Tan |
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Institution: | (1) Department of Chemistry, Fudan University, Shanghai, 200433, China;(2) Institutes of Biomedical Sciences, Fudan University, Shanghai, 200433, China;(3) Department of Modern Physics, University of Science and Technology of China, Hefei, 230026, China; |
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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. |
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