Inactivation of sGC in living cells proceeds without heme loss and involves heterodimer dissociation as a common step.

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Nitric oxide (NO) activates sGC for cGMP production, but in disease sGC becomes insensitive toward NO activation. What changes occur to sGC during its inactivation in cells is not clear.We utilized HEK293 cells expressing sGC proteins to study the changes that occur regarding its heme content, heterodimer status, and sGCβ protein partners when the cells were given oxidant ODQ or the NO donor NOC12 to inactivate sGC. Heme content of sGCβ was monitored in live cells through use of a fluorescent-labeled sGCβ construct, while sGC heterodimer status and protein interactions were studied by Western analysis. Experiments with purified proteins were also performed.ODQ- or NOC12-driven inactivation of sGC in HEK293 cells was associated with heme oxidation (by ODQ), S-nitrosation of the sGCβ subunit (by NOC12), sGC heterodimer breakup, and association of the freed sGCβ subunit with cell chaperone Hsp90. These changes occurred without detectable heme loss from the sGCβ reporter construct. Treating a purified ferrous heme-containing sGCβ with ODQ or NOC12 caused it to bind with Hsp90 without showing any heme loss.Oxidative (ODQ) or nitrosative (NOC12) inactivation of cell sGC involves sGC heterodimer dissociation and rearrangement of the sGCβ protein partners without any heme loss from sGCβ. Clarifying what changes do and do not occur to sGC during its inactivation in cells may direct strategies to preserve or recover NO-dependent cGMP signaling in health and disease.

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Authors: Yue Dai, Dennis J Stuehr