GAPDH Delivers Heme to Soluble Guanylyl Cyclase.

Soluble guanylyl cyclase (sGC) is a key component of nitric oxide (NO)-cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) displays properties of a heme chaperone for inducible NO synthase, we investigated here whether heme delivery to apo-sGCβ involves GAPDH. We utilized an sGCβ reporter construct, tetra-Cys sGCβ (TC-sGCb), 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 either a wild-type GAPDH or a GAPDH variant defective in heme binding recovers the heme delivery. We also studied interaction between GAPDH and sGCβ in cells and their complex formation and potential heme transfer using purified proteins. We found that (i) heme delivery to apo-sGCβ correlates with cellular GAPDH expression levels and depends on the ability of GAPDH to bind intracellular heme; (ii) apo-sGCβ associates with GAPDH in cells and dissociates when heme binds sGCβ; (iii) the purified GAPDH-heme complex binds to apo-sGCβ and transfers its heme to sGCβ. On the basis of these results, we propose a model whereby GAPDH obtains mitochondrial heme and then forms a complex with apo-sGCβ to accomplish heme delivery to sGCβ. Our findings illuminate a critical step in sGC maturation and uncover an additional mechanism that regulates its activity in health and disease.