Spectroscopic insights into axial ligation and active-site H-bonding in substrate-bound human heme oxygenase-2

Heme oxygenases (HOs) are monooxygenases that catalyze the first step in heme degradation, converting heme to biliverdin with concomitant release of Fe(II) and CO from the porphyrin macrocycle. Two heme oxygenase isoforms, HO-1 and HO-2, exist that differ in several ways, including a complete lack of Cys residues in HO-1 and the presence of three Cys residues as part of heme-regulatory motifs (HRMs) in HO-2. HRMs in other heme proteins are thought to directly bind heme, or to otherwise regulate protein stability or activity; however, it is not currently known how the HRMs exert these effects on HO-2 function. To better understand the properties of this vital enzyme and to elucidate possible roles of its HRMs, various forms of HO-2 possessing distinct alterations to the HRMs were prepared. In this study, variants with Cys265 in a thiol form are compared with those with this residue in an oxidized (part of a disulfide bond or existing as a sulfenate moiety) form. Absorption and magnetic circular dichroism spectroscopic data of these HO-2 variants clearly demonstrate that a new low-spin Fe(III) heme species characteristic of thiolate ligation is formed when Cys265 is reduced. Additionally, absorption, magnetic circular dichroism, and resonance Raman data collected at different temperatures reveal an intriguing temperature dependence of the iron spin state in the heme–HO-2 complex. These findings are consistent with the presence of a hydrogen-bonding network at the heme’s distal side within the active site of HO-2 with potentially significant differences from that observed in HO-1.     

Interaction of heme oxygenase-2 with nitric oxide donors. Is the oxygenase an intracellular 'sink' for NO?

Heme oxygenase-2 (HO-2) is the constitutive cognate of the heat-shock protein-32 family of proteins. These proteins catalyze oxidative cleavage of heme to CO and biliverdin, and release Fe. HO-2 is a hemoprotein and binds heme at heme regulatory motifs (HRMs) with a conserved Cys-Pro pair; two copies of HRM are present in HO-2 (Cys264 and Cys281). The HO-2 HRMs are not present in HO-1 and are not involved in HO-2 catalytic activity. Optical CD, and spectral and activity analyses were used to examine reactivity of HO isozymes with NO species produced by NO donors. Purified Escherichia coli-expressed HO preparations, wild-type HO-2, Cys264/Cys281 --> Ala/Ala HO-2-mutant (HO-2-mut) and HO-1 preparations were used. A type II change (red shift) of the Soret band (405 nm --> 413-419 nm) was observed when wild-type HO-2 was treated with sodium nitroprusside (SNP), S-nitroglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine (SIN-1); the NO scavenger, hydroxocobalamin (HCB) prevented the shift. Only SIN-1, which produces peroxynitrite by generating both NO and superoxide anion, decreased the Soret region absorption and the pyridine hemochromogen spectrum of HO-2; superoxide dismutase (SOD) blocked the decrease. Binding of heme to HO-2 protein was required for shift and/or decrease in absorption of the Soret band. NO donors significantly inhibited HO-2 activity, with SNP being the most potent inhibitor (> 40%). Again, trapping NO with HCB blocked HO-2 inactivation. HO-1 and HO-2-mut were not inactivated by NO donors. CD data suggest that the decrease in HO-2 activity was not related to change by NO species of the secondary structure of HO-2. Western blot analysis suggests that NO donors did not cause HO-1 protein loss and Northern blot analysis of HeLa cells treated with SIN-1 and SNP indicates that, unlike HO-1 mRNA, which is remarkably responsive to the treatments, HO-2 mRNA levels were modestly increased ( approximately two to threefold) by NO donors. The data are consistent with the possibility that NO interaction with HO-2-bound heme effects electronic interactions of residues involved in substrate binding and/or oxygen activation. The findings permit the hypothesis that HO-2 and NO are trans-inhibitors, whereby biological activity of NO is attenuated by interaction with HO-2, serving as an intracellular 'sink' for the heme ligand, and NO inhibits HO-2 catalytic activity. As such, the cellular level of both signaling molecules, CO and NO would be moderated.     

Heme Regulatory Motifs in Heme Oxygenase-2 Form a Thiol/Disulfide Redox Switch That Responds to the Cellular Redox State

Heme oxygenase (HO) catalyzes the rate-limiting step in heme catabolism to generate CO, biliverdin, and free iron. Two isoforms of HO have been identified in mammals: inducible HO-1 and constitutively expressed HO-2. HO-1 and HO-2 share similar physical and kinetic properties but have different physiological roles and tissue distributions. Unlike HO-1, which lacks cysteine residues, HO-2 contains three Cys-Pro signatures, known as heme regulatory motifs (HRMs), which are known to control processes related to iron and oxidative metabolism in organisms from bacteria to humans. In HO-2, the C-terminal HRMs constitute a thiol/disulfide redox switch that regulates affinity of the enzyme for heme (Yi, L., and Ragsdale, S. W. (2007) J. Biol. Chem. 282, 20156–21067). Here, we demonstrate that the thiol/disulfide switch in human HO-2 is physiologically relevant. Its redox potential was measured to be −200 mV, which is near the ambient intracellular redox potential. We expressed HO-2 in bacterial and human cells and measured the redox state of the C-terminal HRMs in growing cells by thiol-trapping experiments using the isotope-coded affinity tag technique. Under normal growth conditions, the HRMs are 60–70% reduced, whereas oxidative stress conditions convert most (86–89%) of the HRMs to the disulfide state. Treatment with reductants converts the HRMs largely (81–87%) to the reduced dithiol state. Thus, the thiol/disulfide switch in HO-2 responds to cellular oxidative stress and reductive conditions, representing a paradigm for how HRMs can integrate heme homeostasis with CO signaling and redox regulation of cellular metabolism.Heme oxygenase (HO³ ; EC 1.14.99.3) 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 (1–5) 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, 9). 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, 13); 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 in HRM2⁴ (12, 14) (Fig. 1). 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).Open in a separate windowFIGURE 1.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, 17) 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, 19), coordination of protein synthesis and heme availability in reticulocytes (20, 21), and controlling iron and heme homeostasis (22–26). 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 and Cys²⁸², 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.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

Structural environment dictates the biological significance of heme-responsive motifs and the role of Hsp90 in the activation of the heme activator protein Hap1

Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins. The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1. To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1. We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90. Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90. These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes. The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence. Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction. It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation. Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Heme oxygenase and heme degradation

The microsomal heme oxygenase system consists of heme oxygenase (HO) and NADPH-cytochrome P450 reductase, and plays a key role in the physiological catabolism of heme which yields biliverdin, carbon monoxide, and iron as the final products. Heme degradation proceeds essentially as a series of autocatalytic oxidation reactions involving heme bound to HO. Large amounts of HO proteins from human and rat can now be prepared in truncated soluble form, and the crystal structures of some HO proteins have been determined. These advances have greatly facilitated the understanding of the mechanisms of individual steps of the HO reaction. HO can be induced in animals by the administration of heme or several other substances; the induction is shown to involve Bach1, a translational repressor. The induced HO is assumed to have cytoprotective effects. An uninducible HO isozyme, HO-2, has been identified, so the authentic HO is now called HO-1. HOs are also widely distributed in invertebrates, higher plants, algae, and bacteria, and function in various ways according to the needs of individual species.     

A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.

Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.     

Crystal structure of human heme oxygenase-1 in a complex with biliverdin

Heme oxygenase oxidatively cleaves heme to biliverdin, leading to the release of iron and CO through a process in which the heme participates both as a cofactor and as a substrate. Here we report the crystal structure of the product, iron-free biliverdin, in a complex with human HO-1 at 2.19 A. Structural comparisons of the human biliverdin-HO-1 structure with its heme complex and the recently published rat HO-1 structure in a complex with the biliverdin-iron chelate [Sugishima, M., Sakamoto, H., Higashimoto, Y., Noguchi, M., and Fukuyama, K. (2003) J. Biol. Chem. 278, 32352-32358] show two major differences. First, in the absence of an Fe-His bond and solvent structure in the active site, the distal and proximal helices relax and adopt an "open" conformation which most likely encourages biliverdin release. Second, iron-free biliverdin occupies a different position and orientation relative to heme and the biliverdin-iron complex. Biliverdin adopts a more linear conformation and moves from the heme site to an internal cavity. These structural results provide insight into the rate-limiting step in HO-1 catalysis, which is product, biliverdin, release.     

Modulation of heme oxygenase in tissue injury and its implication in protection against gastrointestinal diseases.

Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, followed by production of biliverdin, free iron and carbon monoxide (CO). There are three isoforms of HO: HO-1 is highly inducible, whereas HO-2 and HO-3 are constitutively expressed. In addition to heme, a variety of nonheme compounds, including heavy metals, cytokines, endotoxins and heat shock stress are strong inducers of HO-1 expression. Many studies indicated that induction of HO-1 is associated with a protective response due to the removal of free heme, which is shown to be toxic. However, recent studies demonstrated that the expression of HO-1 in response to different inflammatory mediators could contribute in part to the resolution of inflammation and have protective effects on brain, liver, kidney and lung against injuries. These beneficial effects seem to be due to the production of bile pigment biliverdin and bilirubin that is a potent antioxidant, as well as the release of iron and CO. However, there are few studies concerning the relationship between HO-1 and inflammation as well as injury in the gut. Interestingly, a preliminary study implicated that induction of HO-1 expression in a colonic damage model induced by trinitrobenzene sulfonic acid played a critical protective role, indicating that activation of HO-1 could act as a natural defensive mechanism to alleviate inflammation and tissue injury in the gastrointestinal tract.     

Carbon monoxide actuates O(2)-limited heme degradation in the rat brain

The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O(2)-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure (PO(2)). High PO(2) thus hastens CO elimination and toxicity resolution, but with more O(2), CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/PO(2). Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/PO(2) vs. [CO] fails even at hyperbaric PO(2). Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O(2) is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO(2). The latter becomes rate limiting for HO activity even at physiological PO(2), and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O(2) that must be accounted for when evaluating oxidative stress and biological signaling by these gases.     

Crystal structure of heme oxygenase-1 from cyanobacterium Synechocystis sp. PCC 6803 in complex with heme.

Heme oxygenase (HO) catalyzes the oxidative degradation of heme utilizing molecular oxygen and reducing equivalents. In photosynthetic organisms, HO functions in the biosynthesis of such open-chain tetrapyrroles as phyto-chromobilin and phycobilins, which are involved in the signal transduction for light responses and light harvesting for photosynthesis, respectively. We have determined the first crystal structure of a HO-1 from a photosynthetic organism, Synechocystis sp. PCC 6803 (Syn HO-1), in complex with heme at 2.5 A resolution. Heme-Syn HO-1 shares a common folding with other heme-HOs. Although the heme pocket of heme-Syn HO-1 is, for the most part, similar to that of mammalian HO-1, they differ in such features as the flexibility of the distal helix and hydrophobicity. In addition, 2-propanol derived from the crystallization solution occupied the hydrophobic cavity, which is proposed to be a CO trapping site in rat HO-1 that suppresses product inhibition. Although Syn HO-1 and mammalian HO-1 are similar in overall structure and amino acid sequence (57% similarity vs. human HO-1), their molecular surfaces differ in charge distribution. The surfaces of the heme binding sides are both positively charged, but this patch of Syn HO-1 is narrow compared to that of mammalian HO-1. This feature is suited to the selective binding of ferredoxin, the physiological redox partner of Syn HO-1; the molecular size of ferredoxin is approximately 10 kDa whereas the size of NADPH-cytochrome P450 reductase, a reducing partner of mammalian HO-1, is approximately 77 kDa. A docking model of heme-Syn HO-1 and ferredoxin suggests indirect electron transfer from an iron-sulfur cluster in ferredoxin to the heme iron of heme-Syn HO-1.     

Unusual heme binding in the bacterial iron response regulator protein: spectral characterization of heme binding to the heme regulatory motif

We characterized heme binding in the bacterial iron response regulator (Irr) protein, which is a simple heme-regulated protein having a single "heme-regulatory motif", HRM, and plays a key role in the iron homeostasis of a nitrogen-fixing bacterium. The heme titration to wild-type and mutant Irr clearly showed that Irr has two heme binding sites: one of the heme binding sites is in the HRM, where (29)Cys is the axial ligand, and the other one, the secondary heme binding site, is located outside of the HRM. The Raman line for the Fe-S stretching mode observed at 333 cm(-1) unambiguously confirmed heme binding to Cys. The lower frequency of the Fe-S stretching mode corresponds to the weaker Fe-S bond, and the broad Raman line of the Fe-S bond suggests multiple configurations of heme binding. These structural characteristics are definitely different from those of typical hemoproteins. The unusual heme binding in Irr was also evident in the EPR spectra. The characteristic g-values of the 5-coordinate Cys-ligated heme and 6-coordinate His/His-ligated heme were observed, while the multiple configurations of heme binding were also confirmed. Such multiple heme configurations are not encountered for typical hemoproteins where the heme functions as the active center. Therefore, we conclude that heme binding to HRM in the heme-regulated protein, Irr, is quite different from that in conventional hemoproteins but characteristic of heme-regulated proteins using heme as the signaling molecule.     

30 some years of heme oxygenase: from a "molecular wrecking ball" to a "mesmerizing" trigger of cellular events

In the beginning, the microsomal HO system was presumed to be made of one isozymes, now known as HO-1, which was cytP450-dependent; and, was thought to be of physiological significance solely in the context of catalysis of hemoglobin heme to bile pigments and CO. A succession of discoveries including characterization of the system as an independent mono-oxygenase, identification of a second form, called HO-2, free radical quenching activity of bile pigments, analogous function of CO in cell signaling to NO, and characterization of the system as HSP32 cognates has led to such an impressive expansion in the number of reports dealing with the HO system that surpass anyone's expectation. This review is a compilation of certain older findings and recent events that together ensure placement of the HO system in the mainstream research for decades to come.     

Role of Cysteine Residues in Heme Binding to Human Heme Oxygenase-2 Elucidated by Two-dimensional NMR Spectroscopy

Human heme oxygenases 1 and 2 (HO-1 and HO-2) degrade heme in the presence of oxygen and NADPH-cytochrome P450 reductase, producing ferrous iron, CO, and biliverdin. HO-1 is an inducible enzyme, but HO-2 is constitutively expressed in selected tissues and is involved in signaling and regulatory processes. HO-2 has three cysteine residues that have been proposed to modulate the affinity for heme, whereas HO-1 has none. Here we use site-specific mutagenesis and two-dimensional NMR of l-[3-¹³C]cysteine-labeled proteins to determine the redox state of the individual cysteines in HO-2 and assess their roles in binding of heme. The results indicate that in the apoprotein, Cys²⁸² and Cys²⁶⁵ are in the oxidized state, probably in an intramolecular disulfide bond. The addition of a reducing agent converts them to the reduced, free thiol state. Two-dimensional NMR of site-specific mutants reveals that the redox state of Cys²⁶⁵ and Cys²⁸² varies with the presence or absence of other Cys residues, indicating that the microenvironments of the Cys residues are mutually interdependent. Cys²⁶⁵ appears to be in a relatively hydrophilic, oxidizable environment compared with Cys¹²⁷ and Cys²⁸². Chemical shift data indicate that none of the cysteines stably coordinates to the heme iron atom. In the oxidized state of the apoprotein, heme is bound 2.5-fold more tightly than in the reduced state. This small difference in heme affinity between the oxidized and reduced states of the protein is much less than previously reported, suggesting that it is not a significant factor in the physiological regulation of cellular heme levels.