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

Abstract

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 in vitro. 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.

Indoleamine dioxygenase and tryptophan dioxygenase activities are regulated through control of cell heme allocation by nitric oxide

“…This suggests that NO may improve heme availability and its mobility inside the cell and deserves further study. We also found that the NO-driven heme allocations to apo-IDO1 and apo-TDO relied on formation of a GAPDH–heme complex in the cells, and for IDO1 also relied on Hsp90 function, which means that the NO acts through a mechanism that involves the same machinery that cells normally use to deliver heme to these dioxygenases ( 29 ) and to several other hemeproteins during their maturation ( 51 , 52 , 53 , 54 ). Within this context NO could act in several ways.…”

Indoleamine Dioxygenase and Tryptophan Dioxygenase Activities are Regulated through Control of Cell Heme Allocation by Nitric Oxide

“…This suggests that NO may improve heme availability and its mobility inside the cell and deserves further study. We also found that the NO-driven heme allocations to apo-IDO1 and apo-TDO relied on formation of a GAPDH-heme complex in the cells, and for IDO1 also relied on Hsp90 function, which means that the NO acts through a mechanism that involves the same machinery that cells normally use to deliver heme to these dioxygenases (29) and to several other hemeproteins during their maturation (51-54). Within this context NO could act in several ways.…”

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO

“…Due to the physical and redox properties of heme, its transport in cells has long been expected to involve a protein chaperone [ [1] , [2] , [3] , [4] , [5] ]. Recently, the glycolytic enzyme GAPDH was shown to fulfill this role by binding mitochondrial heme and through this feature enabling heme delivery to diverse targets including hemoglobin α, β, and γ, myoglobin, tryptophan dioxygenase (TDO), indoleamine dioxygenase 1 (IDO1), soluble guanylyl cyclase (sGC), NO synthases, and heme oxygenase 2 [ [6] , [7] , [8] , [9] , [10] , [11] , [12] ]. In most cases, the heme insertions into these proteins also required the cell chaperone Hsp90 and its ATPase activity [ 13 ].…”