Nitric Oxide Reductase Activity in Heme–Nonheme Binuclear Engineered Myoglobins through a One-Electron Reduction Cycle
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FeBMbs are structural and functional models of native bacterial nitric oxide reductases (NORs) generated through engineering of myoglobin. These biosynthetic models replicate the heme–nonheme diiron site of NORs and allow substitutions of metal centers and heme cofactors. Here, we provide evidence for multiple NOR turnover in monoformyl-heme-containing FeBMb1 proteins loaded with FeII, CoII, or ZnII metal ions at the FeB site (FeII/CoII/ZnII–FeBMb1(MF-heme)). FTIR detection of the ν(NNO) band of N2O at 2231 cm–1 provides a direct quantitative measurement of the product in solution. A maximum number of turnover is observed with FeII–FeBMb1(MF-heme), but the NOR activity is retained when the FeB site is loaded with ZnII. These data support the viability of a one-electron semireduced pathway for the reduction of NO at binuclear centers in reducing conditions.Keywords:
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The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme‐regulated eIF‐2α kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF‐2α kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent K i of 0.5 μM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro . Our results suggest that there are two distinct types of heme‐binding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.
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Abstract Background The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established. Results The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp) indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp. Conclusion We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes . Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes .
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A native protein in a biological system spontaneously produces large and elegant assemblies via self-assembly or assembly with various biomolecules which provide non-covalent interactions. In this context, the protein plays a key role in construction of a unique supramolecular structure operating as a functional system. Our group has recently highlighted the structure and function of hemoproteins reconstituted with artificially created heme analogs. The heme molecule is a replaceable cofactor of several hemoproteins. Here, we focus on the successive supramolecular protein assemblies driven by heme–heme pocket interactions to afford various examples of protein fibers, networks and three-dimensional clusters in which an artificial heme moiety is introduced onto the surface of a hemoprotein via covalent linkage and the native heme cofactor is removed from the heme pocket. This strategy is found to be useful for constructing hybrid materials with an electrode or with nanoparticles. The new systems described herein are expected to lead to the generation of various biomaterials with functions and characteristic physicochemical properties similar to those of hemoproteins.
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IntroductionHeme is ferrous protoporphyrin-IX that is the prosthetic group of hemoproteins, such as hemoglobin, myoglobin and cytochromes that are of vital importance.In contrast, "free heme", a protein-unbound heme, that is either just synthesized but yet not incorporated into hemoproteins, or that is released from hemoprotein under oxidative conditions, is highly toxic, since it catalyzes the production of reactive oxygen species (ROS).Thus, heme proteins and free heme have an important relationship with oxidative stress.In order to cope with this problem, the body is equipped with various defense mechanism(s) against an excessive amount of "free heme" concentrations.Heme oxygenase (HO) is one of the key players in the defense mechanism, and plays a fundamental role against the freeheme mediated oxidative process.The rate-limiting enzyme in heme catabolism, heme oxygenase-1 (HO-1), is induced by not only its substrate heme but also oxidative stress resulting from I/R injury.Heme oxygenase-1 induction leads to increased heme breakdown, resulting in the production of iron, carbon monoxide (CO), and biliverdin IXα, which is subsequently reduced to bilirubin IXα by biliverdin reductase.Recently, large numbers of reports including ours have emerged suggesting heme proteins, HO, and its substrates such as CO, biliverdin IXα, and bilirubin IXα play important roles in pathophysiology and therapeutic implications.Here we summurize these evidences to clarify the relationship among heme proteins, HO-1, and oxidative stress. Synthesis and degradation of heme protein 1Heme is the prosthetic group of all heme proteins such as hemoglobin, myoglobin, cytochrome, catalase, peroxidases, nitric oxide synthase, prostaglandin synthase, and certain transcription factors.Heme is an essential molecule in all aerobic cells and plays a crucial role in physiological, pharmacological, and toxicological reactions, as well as cell differentiation and other functions.However, free heme, namely protein-unbound heme, can be toxic to cells because it results in the production of reactive oxygen species and www.intechopen.
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Abstract Supramolecular hemoprotein assemblies via hemeheme pocket interaction were prepared by synthetic heme dimers containing a linker with charged amino acids and apohemoprotein disulfide dimers. The mixture of the negatively charged heme dimer and the apomyoglobin dimer provides heterotropic fibrous hemoprotein assemblies, which were characterized by size‐exclusion chromatography (SEC) and atomic force microscopy (AFM).
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We demonstrate a new strategy for the construction of supramolecular hemoprotein assemblies. A synthetic heme was selectively introduced onto the surface Cys residue of the cytochrome b562 single mutant (H63C) through a thioether bond. After removal of the native heme of the H63C mutant by acid denaturation followed by neutralization, the externally attached heme on the apoprotein surface was inserted into the vacant heme pocket of the other apoprotein. Therefore, the interprotein heme−heme pocket interaction produces a unique submicrometer-sized linear hemoprotein fiber, determined by size exclusion chromatography and atomic force microscopy. This methodology should be widely applicable to the creation of new nanobiomaterials based on a functional hemoprotein.
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Heme is a non-protein autoantigen which is ubiquitous in vivo, primarily complexed in various hemoproteins or bound to specialized carrier molecules. Nevertheless, heme is able to stimulate a high frequency of CD4+, class II-restricted T cells, freshly explanted from unprimed mice, to proliferate in vitro. In this study, we show that heme incorporated into various species of mammalian cytochrome c (cyt c), including murine cyt c, represents a facultative cryptic determinant, able to be recalled only at high doses of native cyt c. By contrast, avian cyt c is of comparable antigenicity to free heme. Artificially denatured carboxymethylated (CM) mammalian cyt c exhibited greatly increased antigenicity, comparable to that of heme and avian cyt c, indicating that the crypticity of heme in native mammalian cyt c is due to the resistance of the native conformation of this molecule to antigen processing within murine antigen-presenting cells. Thus, tolerance to the heme group of at least some hemoproteins, may be maintained by the crypticity of the heme, rather than by deletion of heme-reactive T cells. Given the high frequency of heme-reactive T cells in unprimed mice, these findings suggest that heme may become an important modulator during an inflammatory response.
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCovalent bonding of the prosthetic heme to protein: a potential mechanism for the suicide inactivation or activation of hemoproteinsYoichi Osawa and Lance R. PohlCite this: Chem. Res. Toxicol. 1989, 2, 3, 131–141Publication Date (Print):May 1, 1989Publication History Published online1 May 2002Published inissue 1 May 1989https://pubs.acs.org/doi/10.1021/tx00009a001https://doi.org/10.1021/tx00009a001research-articleACS PublicationsRequest reuse permissionsArticle Views239Altmetric-Citations71LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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Heme is an iron-containing prosthetic group necessary for the function of several proteins termed "hemoproteins". Erythrocytes contain most of the body's heme in the form of hemoglobin and contain high concentrations of free heme. In non-erythroid cells, where cytosolic heme concentrations are 2-3 orders of magnitude lower, heme plays an essential and often overlooked role in a variety of cellular processes. Indeed, hemoproteins are found in almost every subcellular compartment and are integral in cellular operations such as oxidative phosphorylation, amino acid metabolism, xenobiotic metabolism, and transcriptional regulation. Growing evidence reveals the participation of heme in dynamic processes such as circadian rhythms, NO signaling, and the modulation of enzyme activity. This dynamic view of heme biology uncovers exciting possibilities as to how hemoproteins may participate in a range of physiologic systems. Here we discuss how heme is regulated at the level of its synthesis, availability, redox state, transport, and degradation and highlight the implications for cellular function and whole organism physiology.
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