Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Activation of heme-regulated eukaryotic initiation factor 2alpha kinase by nitric oxide is induced by the formation of a five-coordinate NO-heme complex: optical absorption, electron spin resonance, and resonance raman spectral studies

Heme-regulated eukaryotic initiation factor 2alpha kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains unclear. In the present study, we utilize in vitro kinase assays, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2alpha kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. Interestingly, optical absorption, ESR, and resonance Raman spectra of the Fe(II)-NO complex were characteristic of five-coordinate NO-heme. Spectral findings on the coordination structure of full-length HRI were distinct from those obtained for the isolated N-terminal heme-binding domain. Specifically, six-coordinate NO-Fe(II)-His was observed but not Cys-Fe(III) coordination. It is suggested that significant conformational change(s) in the protein induced by NO binding to the heme lead to HRI activation. We discuss the role of NO and heme in catalysis by HRI, focusing on heme-based sensor proteins.     

Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli. Optical absorption, magnetic circular dichroism, circular dichroism, and redox potentials.

On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand. In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex. However, the Fe(III) complexes of these mutants are six-coordinated low-spin, like the wild-type enzyme. The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95. To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein. CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme. The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were considerably lower than that of the wild-type enzyme (+70 mV) at -1, -26, and -122 mV vs. SHE, respectively. Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme.     

SOUL in mouse eyes is a new hexameric heme-binding protein with characteristic optical absorption, resonance Raman spectral, and heme-binding properties

SOUL is specifically expressed in the retina and pineal gland and displays more than 40% sequence homology with p22HBP, a heme protein ubiquitously expressed in numerous tissues. SOUL was purified as a dimer in the absence of heme from the Escherichia coli expression system but displayed a hexameric structure upon heme binding. Heme-bound SOUL displayed optical absorption and resonance Raman spectra typical of 6-coordinate low-spin heme protein, with one heme per monomeric unit for both the Fe(III) and Fe(II) complexes. Spectral data additionally suggest that one of the axial ligands of the Fe(III) heme complex is His. Mutation of His42 (the only His of SOUL) to Ala resulted in loss of heme binding, confirming that this residue is an axial ligand of SOUL. The K(d) value of heme for SOUL was estimated as 4.8 x 10(-9) M from the association and dissociation rate constants, suggesting high binding affinity. On the other hand, p22HBP was obtained as a monomer containing one heme per subunit, with a K(d) value of 2.1 x 10(-11) M. Spectra of heme-bound p22HBP were different from those of SOUL but similar to those of heme-bound bovine serum albumin in which heme bound to a hydrophobic cavity with no specific axial ligand coordination. Therefore, the heme-binding properties and coordination structure of SOUL are distinct from those of p22HBP, despite high sequence homology. The physiological role of the new heme-binding protein, SOUL, is further discussed in this report.     

Characterization of heme-regulated eIF2alpha kinase: roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition

Heme-regulated eIF2alpha kinase [heme-regulated inhibitor (HRI)] plays a critical role in the regulation of protein synthesis by heme iron. The kinase active site is located in the C-terminal domain, whereas the N-terminal domain is suggested to regulate catalysis in response to heme binding. Here, we found that the rate of dissociation for Fe(III)-protoporphyrin IX was much higher for full-length HRI (1.5 x 10(-)(3) s(-)(1)) than for myoglobin (8.4 x 10(-)(7) s(-)(1)) or the alpha-subunit of hemoglobin (7.1 x 10(-)(6) s(-)(1)), demonstrating the heme-sensing character of HRI. Because the role of the N-terminal domain in the structure and catalysis of HRI has not been clear, we generated N-terminal truncated mutants of HRI and examined their oligomeric state, heme binding, axial ligands, substrate interactions, and inhibition by heme derivatives. Multiangle light scattering indicated that the full-length enzyme is a hexamer, whereas truncated mutants (truncations of residues 1-127 and 1-145) are mainly trimers. In addition, we found that one molecule of heme is bound to the full-length and truncated mutant proteins. Optical absorption and electron spin resonance spectra suggested that Cys and water/OH(-) are the heme axial ligands in the N-terminal domain-truncated mutant complex. We also found that HRI has a moderate affinity for heme, allowing it to sense the heme concentration in the cell. Study of the kinetics showed that the HRI kinase reaction follows classical Michaelis-Menten kinetics with respect to ATP but sigmoidal kinetics and positive cooperativity between subunits with respect to the protein substrate (eIF2alpha). Removal of the N-terminal domain decreased this cooperativity between subunits and affected the other kinetic parameters including inhibition by Fe(III)-protoporphyrin IX, Fe(II)-protoporphyrin IX, and protoporphyrin IX. Finally, we found that HRI is inhibited by bilirubin at physiological/pathological levels (IC(50) = 20 microM). The roles of the N-terminal domain and the binding of heme in the structural and functional properties of HRI are discussed.     

Circadian rhythms in murine pups develop in absence of a functional maternal circadian clock

A genetic approach was used to investigate whether the emergence of circadian rhythms in murine pups is dependent on a functional maternal clock. Arrhythmic females bearing either the mPer1Brdm1/Per2Brdm1 or mPer2Brdm1/Cry1-/- double-mutant genotype were crossed with wild-type males under constant darkness. The heterozygous offspring have the genetic constitution for a functional circadian clock. Individual pups born to arrhythmic mPer1Brdm1/Per2Brdm1 and mPer2Brdm1/Cry1-/- mothers in constant darkness without external zeitgeber developed normal circadian rhythms, but their clocks were less synchronized to each other compared to wild-type animals. These findings indicate that development of circadian rhythms does not depend on a functional circadian clock in maternal tissue, extending previous findings obtained from pups born to SCN-lesioned mothers.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

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.