Heme oxygenase (HO3
; EC 22.214.171.124) catalyzes the O2
- and NADPH-dependent conversion of heme to biliverdin, carbon monoxide (CO), and iron in a reaction that is coupled to cytochrome P450 reductase. Then, biliverdin reductase catalyzes the NADPH-dependent reduction of biliverdin to the antioxidant bilirubin. Several recent reviews on HO (1
) and biliverdin reductase (6
) are available. HO is present in organisms from bacteria to eukaryotes and, as the only known enzyme that can degrade heme, plays a critical role in heme and iron homeostasis.
There are two major HO isoforms in mammals: inducible HO-1, which is ancient and widely distributed among organisms from bacteria to man, and constitutively expressed HO-2, which emerged 250 million years ago with the amniotes (7
). HO-1 is found in most tissues and is highly expressed in spleen and liver (8
). Conversely, HO-2 has a narrow tissue distribution, exhibiting high expression levels in the brain, testes, and carotid body (8
). Both HO-1 and HO-2 catalyze the NADPH- and cytochrome P450 reductase-dependent degradation of heme to CO, iron, and biliverdin, which is quickly reduced to bilirubin in the presence of biliverdin reductase (10
). Controlling cellular heme concentrations is crucial because heme is required as a prosthetic group by regulatory and redox proteins, yet concentrations higher than 1 μm
free heme are toxic (11
). Thus, as the only mammalian proteins known to degrade heme, HOs play a key role in cellular heme homeostasis; furthermore, in vitro
and in vivo
studies of cellular and tissue injuries, such as oxidative stress and hemin-induced cytotoxicity, indicate that HO is cytoprotective (9
HO-1 and HO-2 share high sequence and three-dimensional structural homology in their core domains (12
); however, their sequences diverge near their C termini, in which HO-2 contains two conserved heme regulatory motifs (HRMs), involving Cys265
in HRM1 and Cys282
) (). It was shown recently that the HRMs in HO-2 do not bind heme per se
but instead form a reversible thiol/disulfide redox switch that indirectly regulates the affinity of HO-2 for heme (14
). However, for this redox switch to have any physiological consequence, the midpoint redox potential of the thiol/disulfide couple must be near the ambient intracellular redox potential, estimated to range from −170 to −250 mV (15
Major structural regions in HO-1 and HO-2. His25 in HO-1 or His45 in HO-2 is the heme-binding ligand.
The HRM has been proposed to constitute a heme-binding site (16
) that regulates key metabolic processes from bacteria to humans. The HRM consists of a conserved Cys-Pro core sequence that is usually flanked at the N terminus by basic amino acids and at the C terminus by a hydrophobic residue. HRM/heme interactions have been proposed to regulate the activity and/or stability of proteins that play central roles in respiration and oxidative damage (18
), coordination of protein synthesis and heme availability in reticulocytes (20
), and controlling iron and heme homeostasis (22
). An important component of the last process is HO-2.
Here, we demonstrate that the C-terminal HRMs, which form a thiol/disulfide redox switch between Cys265
, exhibit a redox potential that falls well within the ambient cellular redox potential. By expressing HO-2 in bacterial and human cells and trapping the thiols using the isotope-coded affinity tag (ICAT) technique, it was shown that the redox state of the C-terminal HRMs in growing cells responds to the cellular redox state. The disulfide state is favored under oxidative conditions, and the dithiol state is predominant under reducing conditions. Thus, the HRMs act as a molecular rheostat that responds to the ambient intracellular redox potential and, based on earlier studies (14
), controls activity of HO-2 by regulating heme binding to the enzyme.