GAPDH delivers heme to soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a key component of NO?cGMP signaling in mammals. Although heme must bind in the sGC ?1 subunit (sGC?) for sGC to function, how heme is delivered to sGC? remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGC? involves GAPDH. We utilized an sGC? reporter construct, tetra-Cys sGC?, whose heme insertion can be followed by fluorescence quenching in live cells, assessed how lowering cell GAPDH expression impacts heme delivery, and examined whether expressing WT GAPDH or a GAPDH variant defective in heme binding recovers heme delivery. We also studied interaction between GAPDH and sGC? in cells and their complex formation and potential heme transfer using purified proteins. We found that heme delivery to apo-sGC? correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme, that apo-sGC? associates with GAPDH in cells and dissociates when heme binds sGC?, and that the purified GAPDH?heme complex binds to apo-sGC? and transfers its heme to sGC?. On the basis of these results, we propose a model where GAPDH obtains mitochondrial heme and then forms a complex with apo-sGC? to accomplish heme delivery to sGC?. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.