Abstract
Heme oxygenases (HOs) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms of HO: the inducible HO-1, which is upregulated in response to excess heme and other stressors, and the constitutive HO-2. Much is known about the regulation and physiological function of HO-1, whereas comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. Rather, we discovered a novel role for HO-2 in the binding and buffering of heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor that controls heme bioavailability. When heme is in excess, HO-1 is induced, and both HO-2 and HO-1 can provide protection from heme toxicity via enzymatic degradation. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with labile heme being oxidized, thereby providing new insights into heme trafficking and signaling.
Highlights
Biosynthetic and catabolic enzymes are known and well understood [11, 15]
To determine how active heme oxygenases (HO)-2 is in HEK293 cells, we first sought to determine the amount of cytosolic labile heme (LH)
Heme sensor 1 (HS1) is a tri-domain construct consisting of a heme-binding domain, cytochrome b562 (Cyt b562), an enhanced green fluorescent protein whose emission is quenched by heme, and a red fluorescent protein whose emission is relatively unaffected by heme [63]
Summary
Heme oxygenase-2 (HO-2) binds and buffers labile ferric heme in human embryonic kidney cells. Total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or overexpressed HO-2, as well as various HO-2 mutant alleles, we found that endogenous heme is too limiting a substrate to observe HO-2-dependent heme degradation. HO-2 may act within a network of LH complexes and serve as an access point for heme distribution, possibly to the ER Such a model would require that LH is largely oxidized and exchangeable with HO-2 and would predict that perturbations in HO-2 expression will alter LH or buffered-free heme. In the present report, using the model human cell line, HEK293 cells, we sought to determine if endogenous LH is sufficiently accessible and abundant for changes in HO-2 expression to impact heme degradation and establish the oxidation state of LH. Our findings force us to rethink the physiological role of constitutive HO-2 in cell types that have buffered-free heme levels well below its heme KM values
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