Heme delivery to heme oxygenase-2 involves glyceraldehyde-3-phosphate dehydrogenase.

Heme regulatory motifs (HRMs) are found in a variety of proteins with diverse biological functions. In heme oxygenase-2 (HO2), heme binds to the HRMs and is readily transferred to the catalytic site in the core of the protein. To further define this heme transfer mechanism, we evaluated the ability of GAPDH, a known heme chaperone, to transfer heme to the HRMs and/or the catalytic core of HO2. Our results indicate GAPDH and HO2 form a complex <i>in vitro.</i> We have followed heme insertion at both sites by fluorescence quenching in HEK293 cells with HO2 reporter constructs. Upon mutation of residues essential for heme binding at each site in our reporter construct, we found that HO2 binds heme at the core and the HRMs in live cells and that heme delivery to HO2 is dependent on the presence of GAPDH that is competent for heme binding. In sum, GAPDH is involved in heme delivery to HO2 but, surprisingly, not to a specific site on HO2. Our results thus emphasize the importance of heme binding to both the core and the HRMs and the interplay of HO2 with the heme pool via GAPDH to maintain cellular heme homeostasis.