Nitric oxide (NO) signaling often relies on it activating cGMP production by the heterodimeric enzyme soluble guanylyl cyclase (sGC). To mature to function an sGCβ subunit must first incorporate heme and then form a heterodimer with a partner α subunit. Our previous studies in cells showed that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) supplies heme to the apo-sGCβ subunit, which is complexed with the cell chaperone Hsp90. Through its ATP hydrolysis, Hsp90 then promotes heme insertion into apo-sGCβ and consequent formation of a functional heterodimer. NO at physiologic levels somehow stimulates cell heme allocation into apo-sGCβ by this process. To gain insight we utilized purified apo-sGCβ and GAPDH reporter proteins whose heme contents can be followed by fluorescence and determined the impact of Hsp90 and NO on heme transfer between them. Results show that heme transfer out of GAPDH and into apo-sGCβ is tightly coupled in all circumstances and is limited by the ability of the apo-sGCβ to incorporate the heme, which in turn relies on a ferric to ferrous heme transition taking place inside the sGCβ. Hsp90 can influence the heme transfer kinetics in a negative or positive manner through its conformational effects on apo-sGCβ, while NO speeds heme transfer by binding to the heme iron and thus speeding heme dissociation from GAPDH. Our findings provide new mechanistic understanding of sGC maturation and how Hsp90 and NO combine to dynamically regulate heme incorporation for sGC heterodimer formation and consequent cGMP production in biological settings.