Soluble guanylyl cyclase maturation in cells depends on reduction of the incoming heme by a cysteine in the enzyme's beta subunit.

Nitric oxide (NO) activates cGMP production by soluble guanylyl cyclase (sGC). To do so, an sGCβ subunit must incorporate heme and form a heterodimer with a partner α subunit. In cells, heme-free (apo) sGCβ obtains heme from glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We recently found that transfer of ferric heme from purified GAPDH to apo-sGCβ was accompanied by heme reduction to ferrous within sGCβ, implicating redox involvement of its Cys residues. Here, we utilized Ser substitutions to probe three conserved, redox active Cys in sGCβ (C78, 122, and 214). Proteins were studied in live HEK293 cells and in purified form. In cells, C78S sGCβ was unique among the variants in being unable to incorporate either added ferric heme or mitochondrially-generated heme, but it did incorporate added ferrous heme-NO and consequently formed an active sGC heterodimer. An internal Cys oxidation event occurred in the wild-type, C122S, and C214S apo-sGCβ proteins during their uptake of ferric heme from GAPDH but did not occur in C78S apo-sGCβ. We conclude: (i) Mitochondrial heme reaching apo-sGCβ in cells is ferric under normal culture conditions. (ii) C78 in the sGCβ subunit provides a reducing equivalent to the incoming ferric heme to reduce it to ferrous and this enables stable heme incorporation. (iii) No other Cys residues in sGCβ or cell thiols can replace the role of C78. Having a protein's heme incorporation rely on an internal Cys redox event is unusual and suggests how sGC maturation may be linked to the thiol redox status in cells.