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  • matrix metalloproteinases Molecular modeling of the sGC H NO

    2021-10-19

    Molecular modeling of the sGC H-NOX domain has placed the β1-domain Cys, β1-C78 and β1-C122, close to the heme binding pocket; specifically, the β1-C78 is placed diametrically opposite to β1-H105, the residue responsible for coordinating the heme iron [42,43]. β1-C78 is enclosed in a highly matrix metalloproteinases pocket surrounded by 3 phenylalanines and a valine [42,43], and β1-C122 is a solvent-exposed, partially buried residue farther away from the heme binding pocket [42,43]. Molecular modeling suggests that the identified Cys are not important to sGC regulation, but in-vitro and in-vivo studies have shown otherwise. Further work is necessary to understand the key interactions between the cysteines and the heme iron. Additionally, the biochemical counterpart to S-nitrosation, namely de-S-nitrosation, can modulate sGC function. Thioredoxin-1 (Trx-1) has been specifically implicated as the enzyme driving sGC de-S-nitrosation [44]. Overexpression of Trx-1 and increased cGMP production demonstrated significant enhancement of NO-stimulated sGC activity while pharmacological inhibition of Trx-1 activity by 1-chloro-2,4-dinitrobenzene (DNCB) led to a significant decrease [44]. Trx-1 interaction with sGC to enhance NO-stimulated sGC activity appears to be brought about by the formation of a mixed disulfide bond between sGC and Trx-1 at Trx-1-C73 and α1-C609 [44]. The sGC α1-C609S mutant was shown to be resistant to sGC desensitization, with overexpression of Trx-1 failing to enhance cGMP production [44]. Molecular modeling of the sGC-Trx-1 complex shows Trx-1 interacting at a distance from the heme binding site [44]. How this distant interaction regulates sGC desensitization remains to be identified. Of note, cytochrome b5 reductase 3 (Cyb5R3) is also shown to interact with sGC [45]. Direct Cyb5R3 interaction with sGC reduces oxidized sGC at its heme iron, essential for sensitizing sGC to NO [45]. While NO binding to the ferrous heme could lead to S-nitrosation of key cysteines in the β1 subunit of sGC and desensitize sGC to NO [43], it remains to be seen if Cyb5R3 can also directly or indirectly modulate cysteine redox state during oxidative stress or affect post-translational S-nitrosation in any capacity [45].
    Heme regulation of sGC The redox state of the heme moiety within sGC is crucial for proper NO signaling and has become a focal point of regulation in many cell states. Heme iron in the reduced state (Fe2+) is necessary for NO binding [46]. In the oxidized state (Fe3+), the heme iron of sGC becomes desensitized to available NO [47], leading to a decrease in cGMP production necessary for activation of downstream proteins and pathways [47]. In the earliest studies examining redox control of sGC, oxidation of the heme iron in sGC was observed to occur by exogenous agents, ferricyanide (FeCN) and methylene blue, resulting in the deactivation of sGC [48]. sGC specific inhibitor NS 2028 was also able to inhibit S-nitroso-glutathione stimulated sGC activity [49]. While NS 2028 is proposed to oxidize the heme iron in sGC, it may also change the coordination of the heme iron to inhibit its activity [49,50]. A flavoprotein-containing NADPH oxidoreductase restored NO signaling following oxidation of the heme iron by FeCN and 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) [51]. Subsequently, Cyb5R3, also known as methemoglobin reductase, was found to be a critical protein in maintaining the reduced state of the heme iron [45]. Excess heme oxidation or mutated, nonfunctional Cyb5R3 led to a significant loss in the vasodilatory properties of sGC [45]. Cyb5R3 was found to be directly responsible for reducing oxidized sGC, promoting the signaling pathway, and allowing for proper vasodilation in blood vessels [45]. Hydrogen sulfide, a signaling molecule with antioxidant effects, was found to also reduce the heme iron, allowing for proper signaling [52]. In the presence of oxidative stress, hydrogen sulfide partially restored sensitivity of sGC to NO [52]. Specifically, hydrogen sulfide augmented the response of sGC towards DEA/NO, an NO donor, but attenuated the response to the heme-independent activator BAY 58-2667 [52]. An overview of heme oxidants and reductants are shown in Fig. 2.