Heme-binding Domain Research Articles

Induction of CYP3A gene and enzyme activity expression in kidney cell of grass carp (Ctenopharyngodon idellus)

Cytochrome P450 3A(CYP3A) is the major cytochrome P450 responsible for the metabolism of en-dogenous and xenobiotic substrates.Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity,and can lead to the formation of drug residues,which threaten the safety of fishery products.We determined the in vitro effects of rifampicin(RIF) on CYP3A mRNA expression and enzyme activity in a freshwater teleost,the grass carp(Ctenopharyngodon idellus).Recently,a number of CYP3A genes have been isolated from several teleost species.Although CYP3A genes in fish contain multiple paralogs differing in gene expression pattern and tissue distribution,CYP3A enzymes exhibit similar catalytic properties because of their structural similarities.The CYP3A gene and its full-length cDNA of 1 849 bp were cloned from grass carp by RT-PCR.The CYP3A gene has an open reading frame(ORF) of 1 542 bp,a stop codon,a 3'-untranslated regions(3' UTR),and a polyA signal is present in the 3'UTR region.The ORF encodes a 513 amino acid(aa) protein,which has a signal peptide(29aa),two transmembrane helixes(23aa and 23aa),a heme binding domain(HBD,21aa),and six substrate recognition sites(SRS1-6).Multiple alignments showed that the CYP3A amino acid se-quence from grass carp is 60% to 92% similar to other vertebrate CYP3As.CYP3A gene expression after induc-tion by RIF in grass carp kidney cells(CIK) was assayed by quantitative real-time PCR.CYP3A-dependent erythromycin N-demethylase(ERND) activity was determined by spectrophotometry,and CYP3A activity was assessed by measuring the formation of formaldehyde.In the induced group,CYP3A mRNA expression reached a plateau at 8h,while the highest level of CYP3A activity occurred at 10h.The results indicated that CYP3A mRNA expression and enzyme activity were significantly higher than in the control group(P0.05).The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity.In addition,the transcription of level CYP3A was apparently higher than the enzyme activity,implying that induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.

Redox and Light Control the Heme-Sensing Activity of AppA

ABSTRACTThe DNA binding activity of the photosystem-specific repressor PpsR is known to be repressed by the antirepressor AppA. AppA contains a blue-light-absorbing BLUF domain and a heme-binding SCHIC domain that controls the interaction of AppA with PpsR in response to light and heme availability. In this study, we have solved the structure of the SCHIC domain and identified the histidine residue that is critical for heme binding. We also demonstrate that dark-adapted AppA binds heme better than light-excited AppA does and that heme bound to the SCHIC domain significantly reduces the length of the BLUF photocycle. We further show that heme binding to the SCHIC domain is affected by the redox state of a disulfide bridge located in the Cys-rich carboxyl-terminal region. These results demonstrate that light, redox, and heme are integrated inputs that control AppA’s ability to disrupt the DNA binding activity of PpsR.

Synthesis and evaluation of bifunctional sGC regulators

Background Our previous studies identified dicyanocobinamide as a novel sGC regulator that targets the catalytic region and synergistically enhances activation of sGC by NO-independent regulators. As proof-of-concept study, we designed and synthesized a set of bifunctional sGC regulators, which consist of cobinamide analog conjugated to a protoporphyrin IX derivative through linkers of varying length and composition (Figure 1). Results The length and composition of the linker was proved to be crucial for sGC activation. Our results indicate that only hybrid molecules containing a 13-16 atom chain linker benefited from synergistic engagement of both heme-binding region and catalytic domain. The hybrid compounds containing linkers connected through an ester bond were not only more stable, but also were more effective than those connected though an amide. Hybrids with shorter or longer linkers, or with different linker composition, were much less potent and were no more active than the cobyrinic acid component alone. The most effective compound was conjugated through an ester bond, contained a 13 atom chain linker and a triazole group close to the cobyrinate moiety. This compound displayed more than 60 fold activation of purified sGC.

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90% of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99%) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin-agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.

Signal Transduction and Phosphoryl Transfer by a FixL Hybrid Kinase with Low Oxygen Affinity: Importance of the Vicinal PAS Domain and Receiver Aspartate

FixL is a prototype for heme-based sensors, multidomain proteins that typically couple a histidine protein kinase activity to a heme-binding domain for sensing of diatomic gases such as oxygen, carbon monoxide, and nitric oxide. Despite the relatively well-developed understanding of FixL, the importance of some of its domains has been unclear. To explore the impact of domain-domain interactions on oxygen sensing and signal transduction, we characterized and investigated Rhizobium etli hybrid sensor ReFixL. In ReFixL, the core heme-containing PAS domain and kinase region is preceded by an N-terminal PAS domain of unknown function and followed by a C-terminal receiver domain. The latter resembles a target substrate domain that usually occurs independently of the kinase and contains a phosphorylatable aspartate residue. We isolated the full-length ReFixL as a soluble holoprotein with a single heme b cofactor. Despite a low affinity for oxygen (K(d) for O₂ of 738 μM), the kinase activity was completely switched off by O₂ at concentrations well below the K(d). A deletion of the first PAS domain strongly increased the oxygen affinity but essentially prohibited autophosphorylation, although the truncated protein was competent to accept phosphoryl groups in trans. These studies provide new insights into histidyl-aspartyl phosphoryl transfers in two-component systems and suggest that the control of ligand affinity and signal transduction by PAS domains can be direct or indirect.

Architecture of the Soluble Receptor Aer2 Indicates an In-Line Mechanism for PAS and HAMP Domain Signaling

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per–Arnt–Sim) and poly-HAMP (histidine kinase–adenylyl cyclase–methyl-accepting chemotaxis protein–phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iβ strand in a manner predicted to alter PAS–PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS–HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.

Heme Binding Constricts the Conformational Dynamics of the Cytochrome b559′ Heme Binding Cavity

Cytochrome b(559)' is a transmembrane protein formed by homodimerization of the 44-residue PsbF polypeptide and noncovalent binding of a heme cofactor. The PsbF polypeptide can dimerize in the absence and presence of heme. To monitor structural alterations associated with binding of heme to the apo-cytochrome, we analyzed the apo- and holo-cytochrome structure by electron paramagnetic resonance spectroscopy. Spin labeling of amino acids located close to the heme binding domain of the cytochrome revealed that the structure of the heme binding domain is unconstrained in the absence of heme. Heme binding restricts the conformational dynamics of the heme binding domain, resulting in the structurally more constricted holo-cytochrome structure.

PAS Domain Residues and Prosthetic Group Involved in BdlA-Dependent Dispersion Response by Pseudomonas aeruginosa Biofilms

Biofilm dispersion by Pseudomonas aeruginosa in response to environmental cues is dependent on the cytoplasmic BdlA protein harboring two sensory PAS domains and a chemoreceptor domain, TarH. The closest known and previously characterized BdlA homolog is the flavin adenine dinucleotide (FAD)-binding Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Here, we made use of alanine replacement mutagenesis of the BdlA PAS domain residues previously demonstrated to be essential for aerotaxis in Aer to determine whether BdlA is a potential sensory protein. Five substitutions (D14A, N23A, W60A, I109A, and W182A) resulted in a null phenotype for dispersion. One protein, the BdlA protein with the G31A mutation (BdlA-G31A), transmitted a constant signal-on bias as it rendered P. aeruginosa biofilms hyperdispersive. The hyperdispersive phenotype correlated with increased interaction of BdlA-G31A with the phosphodiesterase DipA under biofilm growth conditions, resulting in increased phosphodiesterase activity and reduced biofilm biomass accumulation. We furthermore demonstrate that BdlA is a heme-binding protein. None of the BdlA protein variants analyzed led to a loss of the heme prosthetic group. The N-terminal PASa domain was identified as the heme-binding domain of BdlA, with BdlA-dependent nutrient-induced dispersion requiring the PASa domain. The findings suggest that BdlA plays a role in intracellular sensing of dispersion-inducing conditions and together with DipA forms a regulatory network that modulates an intracellular cyclic d-GMP (c-di-GMP) pool to enable dispersion.

Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O2

Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydomonas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand-binding histidine FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O2] had Soret absorption maxima around 415nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind O2 with moderate affinity, 135 and 222μM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O2-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to regulate gene expression.

Dimerization and Heme Binding Are Conserved in Amphibian and Starfish Homologues of the microRNA Processing Protein DGCR8

Human DiGeorge Critical Region 8 (DGCR8) is an essential microRNA (miRNA) processing factor that is activated via direct interaction with Fe(III) heme. In order for DGCR8 to bind heme, it must dimerize using a dimerization domain embedded within its heme-binding domain (HBD). We previously reported a crystal structure of the dimerization domain from human DGCR8, which demonstrated how dimerization results in the formation of a surface important for association with heme. Here, in an attempt to crystallize the HBD, we search for DGCR8 homologues and show that DGCR8 from Patiria miniata (bat star) also binds heme. The extinction coefficients (ε) of DGCR8-heme complexes are determined; these values are useful for biochemical analyses and allow us to estimate the heme occupancy of DGCR8 proteins. Additionally, we present the crystal structure of the Xenopus laevis dimerization domain. The structure is very similar to that of human DGCR8. Our results indicate that dimerization and heme binding are evolutionarily conserved properties of DGCR8 homologues not only in vertebrates, but also in at least some invertebrates.

Progress in genes related to seed-coat color in soybean

Seed-coat color has changed from black to yellow during natural and artificial selection of cultivated soybean from wild soybean, and it is also an important morphological marker. Therefore, discovering genes related to the soybean seed-coat color will play a very important role in breeding and evolutionary study. Different seed-coat colors caused by deposition of various anthocyanin pigments. Although pigmentation has been well dissected at molecular level in several plant species, the genes controlling natural variation of seed-coat color in soybean remain to be unknown. Genes related to seed-coat color in soybean were discussed in this paper, including 5 genetic loci (I, T, W1, R and O). Locus I is located in a region that riches in chalcone synthase (CHS) genes on chromosome 8. Gene CHS is a multi-gene family with highly conserved sequences in soybean. Locus T located on chromosome 6 has been cloned and verified, which encodes a flavon-oid-3'-hydroxylase. Mutant of F3'H can not interact with the heme-binding domain due to lack of conservative domain GGEK caused by a nucleotide deletion in the coding region of F3'H. Locus R is located between A668-1 and K387-1 on chromosome 9 (linkage group K). This locus may encode a R2R3 MYB transcription factor or a UDP flavonoid 3-O glyco-syltransferase. Locus O is located between Satt207 and Satt493 on chromosome 8 (linkage group A2) and its molecular characteristics has not been characterized. Locus W1 may be a homology of F3'5'H gene.

Cloning of the lanosterol 14-α-demethylase (ERG11) gene in Trichosporon asahii: a possible association between G453R amino acid substitution and azole resistance in T. asahii

Lanosterol 14-α-demethylase ( Erg11 protein; Erg11p ), encoded by the ERG11 gene, is the primary target of azoles. Recently, a change in affinity of this enzyme for azoles has been reported as a resistance mechanism in several fungal species. Trichosporon asahii ( T.asahii) is susceptible to fluconazole (FLC). This report identified the ERG11 gene of T.asahii (NCBI accession; HQ176415). The Erg11p of T.asahii, presumed from the DNA sequence, was closely related to the Erg11p of Cryptococcus neoformans. Furthermore, a FLC-susceptible strain was cultured in medium containing FLC at concentrations from 4.0 to 16μgmL(-1) in order to analyze the development of FLC resistance in T.asahii. The degree of resistance was related to the FLC concentration of the growth medium. One highly resistant strain that was cultured in the medium containing 16μgmL(-1) FLC contained 1 point mutation (G1357C) that caused a single amino acid substitution at G453R. This amino acid is highly conserved among major fungal pathogens, and it is in a region very close to the heme-binding domain, which is characteristic of the cytochrome P450 superfamily, the primary target for the azole class of antifungal agents. This amino acid substitution may have caused the high resistance to azoles in T.asahii.

Mapping Ultra-weak Protein-Protein Interactions between Heme Transporters of Staphylococcus aureus

Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.

Caspases cleave and inhibit the microRNA processing protein DiGeorge Critical Region 8

DGCR8 (DiGeorge Critical Region 8) is an essential microRNA (miRNA) processing protein that recognizes primary transcripts of miRNAs (pri-miRNAs) and triggers their cleavage by the Drosha nuclease. We previously found that Fe(III) heme binds and activates DGCR8. Here we report that in HeLa cells, DGCR8 undergoes two proteolytic events that produce two C-terminal fragments called DGCR8(C1) and DGCR8(C2) , respectively. DGCR8(C2) accumulates during apoptosis and is generated through cleavage by a caspase. The caspase cleavage site is located in the central loop of the heme-binding domain. Cleavage of DGCR8 by caspase-3 in vitro results in loss of the otherwise tightly bound Fe(III) heme cofactor, dissociation of the N- and C-terminal proteolytic fragments, and inhibition of the pri-miRNA processing activity. These results reveal an intrinsic mechanism in the DGCR8 protein that seems to have evolved for regulating miRNA processing via association with Fe(III) heme and proteolytic cleavage by caspases. Decreased expression of miRNAs has been observed in apoptotic cells, and this change was attributed to caspase-mediated cleavage of a down-stream miRNA processing nuclease Dicer. We suggest that both the Drosha and Dicer cleavage steps of the miRNA maturation pathway may be inhibited in apoptosis and other biological processes where caspases are activated.

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.

Impacts of Uranyl Ion on the Structure and Function of Cytochrome <i>b</i><sub>5</sub> His39Ser Mutant

Uranium is toxic to human body with mechanisms not fully understood. The structural and functional consequences of uranyl ions (UO22+) interacting with an axial mutant of cytochrome b5, His39Ser (cyt b5 H39S), were investigated by both spectroscopic and molecular modeling methods. Although slightly disturbs protein folding, UO22+ binding to cyt b5 H39S leads to a decrease of peroxidase activity. A uranyl binding site was further proposed in the heme-binding domain at Glu37 and Glu43. The impacts of UO22+ binding to cyt b5 H39S studied herein provide valuable insights into the toxicity mechanism of UO22+ towards membrane heme proteins.

CO metabolism, sensing, and signaling

CO is a colorless and odorless gas produced by the incomplete combustion of hydrocarbons, both of natural and anthropogenic origin. Several microorganisms, including aerobic and anaerobic bacteria and anaerobic archaea, use exogenous CO as a source of carbon and energy for growth. On the other hand, eukaryotic organisms use endogenous CO, produced during heme degradation, as a neurotransmitter and as a signal molecule. CO sensors act as signal transducers by coupling a "regulatory" heme-binding domain to a "functional" signal transmitter. Although high CO concentrations inhibit generally heme-protein actions, low CO levels can influence several signaling pathways, including those regulated by soluble guanylate cyclase and/or mitogen-activated protein kinases. This review summarizes recent insights into CO metabolism, sensing, and signaling.

Heme-Regulated eIF2α Kinase in Erythropoiesis and Oxidative Stress

Abstract SCI-23 Besides serving as a prosthetic group for hemoglobin, heme also regulates translation by inhibiting the kinase activity of heme-regulated translational inhibitor (HRI). HRI is the only known protein kinase that senses the intracellular heme concentration and does so through its two heme-binding domains. HRI is activated in heme deficiency by autophosphorylation, and phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), which impairs the recycling of eIF2 for further translational initiation and results in cessation of protein synthesis. In this manner, HRI serves as a feedback inhibitor and coordinates the translation of globin mRNAs with the intracellular heme concentration to ensure that globin proteins are not made in excess of the heme available for the formation of hemoglobin. Excess of globin chains or heme is cytotoxic. In the absence of HRI, uncontrolled protein synthesis in heme deficiency results in globin aggregation and precipitation in red blood cells and their precursors. HRI is responsible for the physiological adaptation that produces hypochromic, microcytic erythrocytes in iron deficiency. The expression of HRI was increased during late stages of erythropoiesis with higher expression in Ter119high erythroblasts and reticulocytes, correlating with the active synthesis and regulation of globins at these stages of erythroid differentiation. In addition to inhibiting global protein synthesis, the second important function of eIF2α phosphorylation is to reprogram translation and the subsequent transcription of genes required for stress response. In mammalian cells, translation of the transcription factor ATF4 mRNA is upregulated specifically by eIF2α phosphorylation via upstream open reading frames in the 5’UTR. Activation of the HRI-ATF4 stress response pathway in nucleated erythroid precursors is required for adaptation to acute and chronic oxidative stress. Furthermore, this HRI-dependent ATF4 pathway is also operative and necessary for erythroid differentiation, especially under stress conditions. In chronic iron deficiency, HRI is necessary for adaptive gene expression for erythroid differentiation as well as for iron heme and redox homeostasis. Beyond heme deficiency and oxidative stress, HRI is also activated by osmotic shock and heat shock. HRI deficiency in mice exacerbates erythropoietic protoporphyria and renders β-thalassemia intermedia embryonically lethal. Hri−/− mice also develop ineffective erythropoiesis during iron/heme deficiency and are severely compromised upon phenylhydrazine-induced acute hemolytic anemia. Thus, translational regulation by HRI plays a critical role in the manifestation of red cell diseases in mice and may be a significant modifier of such diseases in humans. Disclosures: No relevant conflicts of interest to declare.

Identification of Residues in the Heme Domain of Soluble Guanylyl Cyclase that are Important for Basal and Stimulated Catalytic Activity

Nitric oxide signals through activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain) to the effector domain (catalytic domain), in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105) of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.