Soluble guanylate cyclase (sGC) heme iron, in its oxidized state (Fe³⁺), is desensitized to NO and limits cGMP production needed for downstream activation of protein kinase G–dependent signaling and blood vessel dilation.

Although reactive oxygen species are known to oxidize the sGC heme iron, the basic mechanism(s) governing sGC heme iron recycling to its NO-sensitive, reduced state remain poorly understood.

Oxidant challenge studies show that vascular smooth muscle cells have an intrinsic ability to reduce oxidized sGC heme iron and form protein–protein complexes between cytochrome b5 reductase 3, also known as methemoglobin reductase, and oxidized sGC. Genetic knockdown and pharmacological inhibition in vascular smooth muscle cells reveal that cytochrome b5 reductase 3 expression and activity is critical for NO-stimulated cGMP production and vasodilation. Mechanistically, we show that cytochrome b5 reductase 3 directly reduces oxidized sGC required for NO sensitization as assessed by biochemical, cellular, and ex vivo assays.

Together, these findings identify new insights into NO–sGC–cGMP signaling and reveal cytochrome b5 reductase 3 as the first identified physiological sGC heme iron reductase in vascular smooth muscle cells, serving as a critical regulator of cGMP production and protein kinase G–dependent signaling.