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1.
BackgroundThe occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements.MethodsTo understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV–vis, resonance Raman, cw-EPR and 2D NMR spectroscopy.ResultsWe observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69 ± 4.37 μM.ConclusionsIt was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme.General significanceUnderstanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.  相似文献   

2.
Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 μM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 μM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.  相似文献   

3.
4.
A b-type heme is conserved in membrane-bound complex II enzymes (SQR, succinate–ubiquinone reductase). The axial ligands for the low spin heme b in Escherichia coli complex II are SdhC His84 and SdhD His71. E. coli SdhD His71 is separated by 10 residues from SdhD Asp82 and Tyr83 which are essential for ubiquinone catalysis. The same His-10x-AspTyr motif dominates in homologous SdhD proteins, except for Saccharomyces cerevisiae where a tyrosine is at the axial position (Tyr-Cys-9x-AspTyr). Nevertheless, the yeast enzyme was suggested to contain a stoichiometric amount of heme, however, with the Cys ligand in the aforementioned motif acting as heme ligand. In this report, the role of Cys residues for heme coordination in the complex II family of enzymes is addressed. Cys was substituted to the SdhD-71 position and the yeast Tyr71Cys72 motif was also recreated. The Cys71 variant retained heme, although it was high spin, while the Tyr71Cys72 mutant lacked heme. Previously the presence of heme in S. cerevisiae was detected by a spectral peak in fumarate-oxidized, dithionite-reduced mitochondria. Here it is shown that this method must be used with caution. Comparison of bovine and yeast mitochondrial membranes shows that fumarate induced reoxidation of cytochromes in both SQR and the bc1 complex (ubiquinol–cytochrome c reductase). Thus, this report raises a concern about the presence of low spin heme b in S. cerevisiae complex II.  相似文献   

5.
Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0–4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions.  相似文献   

6.
The mitochondrial import of 5-aminolevulinate synthase (ALAS), the first enzyme of the mammalian heme biosynthetic pathway, requires the N-terminal presequence. The 49 amino acid presequence transit peptide (psALAS) for murine erythroid ALAS was chemically synthesized, and circular dichroism and (1)H nuclear magnetic resonance (NMR) spectroscopies used to determine structural elements in trifluoroethanol/H(2)O solutions and micellar environments. A well defined amphipathic alpha-helix, spanning L22 to F33, was present in psALAS in 50% trifluoroethanol. Further, a short alpha-helix, defined by A5-L8, was also apparent in the 26 amino acid N-terminus peptide, when its structure was determined in sodium dodecyl sulfate. Heme inhibition of ALAS mitochondrial import has been reported to be mediated through cysteine residues in presequence heme regulatory motifs (HRMs). A UV/visible and (1)H NMR study of hemin and psALAS indicated that a heme-peptide interaction occurs and demonstrates, for the first time, that heme interacts with the HRMs of psALAS.  相似文献   

7.
Recent reports show that heme binds to amyloid β-peptide (Aβ) in the brain of Alzheimer’s disease (AD) patients and forms Aβ–heme complexes, thus leading a pathological feature of AD. However, the important biological relevance to AD etiology, resulting from human Aβ–heme peroxidase formation, was not well characterized. In this study, we used wild-type and mutated human Aβ1–16 peptides and investigated their Aβ–heme peroxidase activities. Our results indicated that both histidine residues (His13, His14) in Aβ1–16 and free histidine enhanced the peroxidase activity of heme, hence His residues were essential in peroxidase activity of Aβ–heme complexes. Moreover, Arg5 was found to be the key residue in making the Aβ1–16–heme complex as a peroxidase. Under oxidative and nitrative stress conditions, the Aβ1–16–heme complexes caused oxidation and nitration of the Aβ Tyr10 residue through promoting peroxidase-like reactions. Therefore, these residues (Arg5, Tyr10 and His) were pivotal in human Aβ–heme peroxidase activity. However, three of these residues (Arg5, Tyr10 and His13) identified in this study are all absent in rodents, where rodent Aβ–heme complex lacks peroxidase activity and it does not show AD, implicating the novel significance of these residues as well as human Aβ–heme peroxidase in the pathology of AD.  相似文献   

8.
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.  相似文献   

9.
Owens CP  Du J  Dawson JH  Goulding CW 《Biochemistry》2012,51(7):1518-1531
The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 μM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.  相似文献   

10.
Catalase-peroxidase function is strictly dependent on a gene-duplicated C-terminal domain. This domain no longer has a functioning active site, but from 25 to 30 Å away it is essential for preventing the coordination of an active site base (His106) to the heme. The mechanisms by which this distant structure supports active site function have not yet been elucidated. Tyr111 is a strictly conserved member of an interdomain H-bonding network that supports the loop connecting the N-terminal B (bearing His106) and C helices. Spectroscopic evaluation of the Tyr111Ala variant of KatG showed a substantial increase in hexa-coordinate low-spin heme, giving it the appearance of a transition between the wild type (primarily high-spin) and the N-terminal domain alone (pure low-spin). Concomitant with the spectral changes was decreased activity compared to the wild type enzyme, suggesting that Tyr111 does have a role in preventing His106 coordination. Substitution of Tyr111 diminishes catalase activity more substantially than peroxidase activity. Such an effect cannot be explained by His106 coordination alone, suggesting that these interdomain interactions may help tune the catalase-peroxidase active site for bifunctionality.  相似文献   

11.
Kindlins are essential for integrin activation in cell systems and do so by working in a cooperative fashion with talin via their direct interaction with integrin β cytoplasmic tails (CTs). Kindlins interact with the membrane-distal NxxY motif, which is distinct from the talin-binding site within the membrane-proximal NxxY motif. The Tyr residues in both motifs can be phosphorylated, and it has been suggested that this modification of the membrane-proximal NxxY motif negatively regulates interaction with the talin head domain. However, the influence of Tyr phosphorylation of the membrane-distal NxxY motif on kindlin binding is unknown. Using mutational analyses and phosphorylated peptides, we show that phosphorylation of the membrane-distal NITY759 motif in the β3 CT disrupts kindlin-2 recognition. Phosphorylation of this membrane-distal Tyr also disables the ability of kindlin-2 to coactivate the integrin. In direct binding studies, peptides corresponding to the non-phosphorylated β3 CT interacted well with kindlin-2, whereas the Tyr759-phosphorylated peptide failed to bind kindlin-2 with measurable affinity. These observations indicate that transitions between the phosphorylated and non-phosphorylated states of the integrin β3 CT determine reactivity with kindlin-2 and govern the role of kindlin-2 in regulating integrin activation.  相似文献   

12.
13.
AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.  相似文献   

14.
Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.  相似文献   

15.
It is well known that the functions of metalloproteins generally originate from their metal‐binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal‐binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc‐binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc‐binding sequence His–Glu–Xaa–Xaa–His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin‐1 and investigated the folding properties of the Trp‐labeled peptides WAHEIAHSLGLFHA (STR‐W1), AWHEIAHSLGLFHA (STR‐W2), AHEIAHSLGWFHA (STR‐W11), and AHEIAHSLGLFHWA (STR‐W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp‐incorporated peptides, STR‐W1 and STR‐W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR‐W14 revealed that the conformation in the C‐terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn‐bound state of stromelysin‐1 in crystal structures. The structural analyses of the qualitative Zn‐binding properties of this motif peptide provide an interesting Zn‐binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc‐binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

16.
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.  相似文献   

17.
We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme. Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer. The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B. The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme. Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme. No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.  相似文献   

18.
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.  相似文献   

19.
Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. From 20 clusters we extracted 16 known and four new interaction motifs. Using quantitative mass spectrometry we pulled down Tyr(P)-specific binding partners for peptides corresponding to the extracted motifs. We confirmed numerous previously known interaction motifs and found 15 new interactions mediated by phosphosites not previously known to bind SH2 or PTB. Remarkably, a novel hydrophobic N-terminal motif ((L/V/I)(L/V/I)pY) was identified and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid binding.  相似文献   

20.
In humans, heme iron is the most abundant iron source, and bacterial pathogens such as Staphylococcus aureus acquire it for growth. IsdB of S. aureus acquires Fe(III)-protoporphyrin IX (heme) from hemoglobin for transfer to IsdC via IsdA. These three cell-wall-anchored Isd (iron-regulated surface determinant) proteins contain conserved NEAT (near iron transport) domains. The purpose of this work was to delineate the mechanism of heme binding and transfer between the NEAT domains of IsdA, IsdB, and IsdC using a combination of structural and spectroscopic studies. X-ray crystal structures of IsdA NEAT domain (IsdA-N1) variants reveal that removing the native heme-iron ligand Tyr166 is compensated for by iron coordination by His83 on the distal side and that no single mutation of distal loop residues is sufficient to perturb the IsdA-heme complex. Also, alternate heme-iron coordination was observed in structures of IsdA-N1 bound to reduced Fe(II)-protoporphyrin IX and Co(III)-protoporphyrin IX. The IsdA-N1 structural data were correlated with heme transfer kinetics from the NEAT domains of IsdB and IsdC. We demonstrated that the NEAT domains transfer heme at rates comparable to full-length proteins. The second-order rate constant for heme transfer from IsdA-N1 was modestly affected (< 2-fold) by the IsdA variants, excluding those at Tyr166. Substituting Tyr166 with Ala or Phe changed the reaction mechanism to one with two observable steps and decreased observed rates > 15-fold (to 100-fold excess IsdC). We propose a heme transfer model wherein NEAT domain complexes pass heme iron directly from an iron-coordinating Tyr of the donor protein to the homologous Tyr residues of the acceptor protein.  相似文献   

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