ABSTRACT
Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46â C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.
Subject(s)
Heme , Myoglobin , Myoglobin/chemistry , Myoglobin/genetics , Myoglobin/metabolism , Catalytic Domain , Heme/chemistry , Kinetics , Protein Conformation , Sulfhydryl CompoundsABSTRACT
The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.
Subject(s)
Metalloproteins , Myoglobin , Myoglobin/chemistry , Heme/chemistry , Oxazines , Nitric Oxide SynthaseABSTRACT
The worldwide spread of COVID-19 continues to impact our lives and has led to unprecedented damage to global health and the economy. This highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We modified a single-domain antibody, SARS-CoV-2 VHH, to the surface of the liposomes. These immunoliposomes demonstrated a good neutralizing ability, but could also carry therapeutic compounds. Furthermore, we used the 2019-nCoV RBD-SD1 protein as an antigen with Lip/cGAMP as the adjuvant to immunize mice. Lip/cGAMP enhanced the immunity well. It was demonstrated that the combination of RBD-SD1 and Lip/cGAMP was an effective preventive vaccine. This work presented potent therapeutic anti-SARS-CoV-2 drugs and an effective vaccine to prevent the spread of COVID-19.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , SARS-CoV-2 , Single-Domain Antibodies , Animals , Mice , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/chemistry , Antibodies, Viral/therapeutic use , COVID-19/therapy , Liposomes/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/therapeutic useABSTRACT
Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.
Subject(s)
Globins , Nerve Tissue Proteins , Binding Sites , Disulfides/chemistry , Globins/chemistry , Globins/genetics , Globins/metabolism , Heme/chemistry , Humans , Ligands , Nerve Tissue Proteins/chemistry , Neuroglobin , X-RaysABSTRACT
Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α1C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity.
Subject(s)
Guanylate Cyclase , Receptors, Cytoplasmic and Nuclear , Catalytic Domain , Guanylate Cyclase/genetics , Guanylate Cyclase/metabolism , Humans , Nitric Oxide/metabolism , Soluble Guanylyl Cyclase/genetics , Soluble Guanylyl Cyclase/metabolismABSTRACT
Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.
Subject(s)
Cell Membrane/metabolism , Myoglobin/chemistry , Myoglobin/metabolism , Animals , Cell Membrane/drug effects , Cell Survival/drug effects , Circular Dichroism , Crystallography, X-Ray , Fluorescein-5-isothiocyanate/chemistry , Humans , Lysine/chemistry , MCF-7 Cells , Mutation , Myoglobin/genetics , Myoglobin/pharmacokinetics , Spectrophotometry, Ultraviolet , Sperm WhaleABSTRACT
A double mutant of human H64M/V71C neuroglobin (Ngb) was engineered, which formed a single thioether bond as that in atypical cytochrome c, whereas the heme distal Met64 was oxidized to both sulfoxide (SO-Met) and sulfone (SO2 -Met). By contrast, no Cys-heme cross-link was formed in V71C Ngb with His64/His96 coordination, as shown by the X-ray crystal structure, which indicates that an open distal site facilitates the activation of heme iron for structural modifications.
Subject(s)
Cytochromes c , Sulfides , Humans , Cytochromes c/genetics , Cytochromes c/metabolism , Heme/chemistry , Neuroglobin/chemistry , Neuroglobin/metabolism , Oxidation-Reduction , Protein EngineeringABSTRACT
The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes-TANK-binding kinase 1-interferon regulating factor 3 (cGAS-STING-TBK1-IRF3) axis is now acknowledged as the major signaling pathway in innate immune responses. However, 2',3'-cGAMP as a STING stimulator is easily recognized and degraded by ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which reduces the effect of tumor immunotherapy and promotes metastatic progression. In this investigation, the structure-based virtual screening strategy was adopted to discover eight candidate compounds containing zinc-binding quinazolin-4(3H)-one scaffold as ENPP1 inhibitors. Subsequently, these novel inhibitors targeting ENPP1 were synthesized and characterized by NMR and high-resolution mass spectra (HRMS). In bioassays, 7-fluoro-2-(((5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)thio)methyl)quina-zolin-4(3H)-one(compound 4e) showed excellent activity against the ENPP1 at the molecular and cellular levels, with IC50 values of 0.188 µM and 0.732 µM, respectively. Additionally, compound 4e had superior selectivity towards metastatic breast cancer cells (4T1) than towards normal cells (LO2 and 293T) in comparison with cisplatin, indicating that compound 4e can potentially be used in metastatic breast cancer therapy. On the other hand, compound 4e upgraded the expression levels of IFN-ß in vivo by preventing the ENPP1 from hydrolyzing the cGAMP to stimulate a more potent innate immune response. Therefore, this compound might be applied to boost antitumor immunity for cancer immunotherapy. Overall, our work provides a strategy for the development of a promising drug candidate targeting ENPP1 for tumor immunotherapy.
Subject(s)
Breast Neoplasms , Membrane Proteins , Female , Humans , Immunotherapy , Interferons , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , Phosphoric Diester Hydrolases/metabolism , PyrophosphatasesABSTRACT
The Nid site coordination microenvironment of a truncated acetyl-coenzyme A synthase has been designed systematically for functional conversion to a Ni-SOD-like enzyme. To this end, the first strategy is to introduce an axial histidine ligand, using mutations F598H, S594H and S594H-GP individually. The resulting three mutants obtained Ni-SOD-like activity successfully, although the catalytic activity was about 10-fold lower than in native Ni-SOD. The second strategy is to mimic the H-bond network in the second sphere coordination microenvironment of the native Ni-SOD. Two mutations based on F598H (EFG-F598H and YGP-F598H) were designed. The successful EFG-F598H exhibited ~3-fold Ni-SOD-like activity of F598H. These designed Ni-SOD-like metalloproteins were characterized by UV/Vis, EPR and Cyclic voltammetry while F598H was also characterized by X-ray protein crystallography. The pH titrations were performed to reveal the source of the two protons required for forming H2O2 in the SOD catalytic reaction. Based on all of the results, a proposed catalytic mechanism for the Ni-SOD-like metalloproteins is presented.
Subject(s)
Metalloproteins , Nickel , Coenzyme A , Hydrogen Peroxide , Metalloproteins/chemistry , Nickel/chemistry , Protons , Superoxide Dismutase/metabolismABSTRACT
Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive heme enzyme for its significant function in cancer immunotherapy. Potent IDO1 inhibitors have been discovered for decades, whereas no clinical drugs are used for cancer treatment up to now. With the goal of developing medically valuable IDO inhibitors, we performed a systematic study of SAR405838 analogs with a spiro-oxindole skeleton in this study. Based on the expression and purification of human IDO1, the inhibitory activity of spiro-oxindole skeleton compounds to IDO1 was evaluated by IC50 and Ki values. The results demonstrated that inhibitor 3 exhibited the highest IDO1 inhibitory activity with IC50 at 7.9 µM among all inhibitors, which is ~six-fold of the positive control (4-PI). Moreover, inhibitor 3 was found to have the most effective inhibition of IDO1 in MCF-7 cancer cells without toxic effects. Molecular docking analysis revealed that the hydrophobic interaction stabilized the binding of inhibitor 3 to the IDO1 active site and made an explanation for the uncompetitive mode of inhibitors. Therefore, this study provides valuable insights into the screen of more potent IDO1 inhibitors for cancer immunotherapy.
Subject(s)
Enzyme Inhibitors , Indoleamine-Pyrrole 2,3,-Dioxygenase , Oxindoles , Spiro Compounds , Enzyme Inhibitors/chemistry , Humans , Immunotherapy , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Indoles , Molecular Docking Simulation , Oxindoles/pharmacology , Spiro Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The ß-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O2 carrier, also exhibits novel ß-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency ((kcat/KM)/kuncat) was determined to be 6.25 × 1010, which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural ß-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the ß-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as ß-lactamases, which broadens the diversity of their catalytic functions.
Subject(s)
Heme , Myoglobin , Myoglobin/chemistry , Heme/chemistry , Protein Conformation , Models, Molecular , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
Tetracyclines are one class of widely used antibiotics. Meanwhile, due to abuse and improper disposal, they are often detected in wastewater, which causes a series of environmental problems and poses a threat to human health and safety. As an efficient and environmentally friendly method, enzymatic catalysis has attracted much attention. In previous studies, we have designed an efficient peroxidase (F43Y/P88W/F138W Mb, termed YWW Mb) based on the protein scaffold of myoglobin (Mb), an O2 carrier, by modifying the heme active center and introducing two Trp residues. In this study, we further applied it to degrade the tetracycline antibiotics. Both UV-Vis and HPLC studies showed that the triple mutant YWW Mb was able to catalyze the degradation of tetracycline, oxytetracycline, doxycycline, and chlortetracycline effectively, with a degradation rate of ~100%, ~98%, ~94%, and ~90%, respectively, within 5 min by using H2O2 as an oxidant. These activities are much higher than those of wild-type Mb and other heme enzymes such as manganese peroxidase. As further analyzed by UPLC-ESI-MS, we identified multiple degradation products and thus proposed possible degradation mechanisms. In addition, the toxicity of the products was analyzed by using in vitro antibacterial experiments of E. coli. Therefore, this study indicates that the engineered heme enzyme has potential applications for environmental remediation by degradation of tetracycline antibiotics.
Subject(s)
Myoglobin , Tetracycline , Humans , Myoglobin/chemistry , Peroxidase , Hydrogen Peroxide , Escherichia coli/genetics , Escherichia coli/metabolism , Peroxidases/chemistry , Anti-Bacterial Agents/pharmacology , Tetracyclines , Heme/chemistryABSTRACT
DNA methylation is an important epigenetic modification. Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET2-5fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure, and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , 5-Methylcytosine/metabolism , Biocatalysis , Catalytic Domain , Crystallography, X-Ray , Cytosine/analogs & derivatives , Cytosine/metabolism , DNA Methylation , Dioxygenases , Humans , Hydrogen Bonding , Mixed Function Oxygenases , Models, Molecular , Oxidation-Reduction , Protein Binding , Substrate SpecificityABSTRACT
The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency (kcat/Km) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-ß-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.
Subject(s)
Coloring Agents/chemistry , Lignin/metabolism , Myoglobin/chemistry , Peroxidase/metabolism , Protein Engineering , Animals , Chromatography, High Pressure Liquid , Color , Guaifenesin/analogs & derivatives , Heme/chemistry , Hydrogen Peroxide/metabolism , Kinetics , Oxidation-Reduction , Polymerization , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Sperm WhaleABSTRACT
Innate immune responses are considered to play crucial roles in the progression of Alzheimer's disease (AD). Recently, immunotherapy is emerging as an innovative and highly conceivable strategy for AD treatment. The cGAMP-STING-IRF3 signaling pathway plays a pivotal role in mediating innate immune responses. In this study, we provide pioneering investigation to find that the STING stimulator, cGAMP, significantly ameliorates cognitive deficits, improves pathological changes, decreases Aß plaque load and reduces neuron apoptosis in APP/PS1 transgenetic mice. The stimulation of cGAMP-STING-IRF3 pathway induces expression of triggering receptor expressed on myeloid cells 2 (TREM2), and the overexpression of TREM2 further decreases Aß deposition and neuron loss while improves AD pathomorphology and cognitive impairment. Additionally, TREM2 regulates microglia polarization from M1 towards M2 phenotype thereby achieves reduction of neuroinflammation in AD. These findings support that the enhancement of TREM2 exerts beneficial effects in ameliorating AD development. Taken together, our results demonstrate that cGAMP is a potential candidate for applications in Alzheimer's disease immunotherapy.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Membrane Glycoproteins/metabolism , Nucleotides, Cyclic/pharmacology , Receptors, Immunologic/metabolism , Alzheimer Disease/pathology , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Cognition Disorders/metabolism , Cognitive Dysfunction/metabolism , Disease Models, Animal , Disease Progression , Interferon Regulatory Factor-3/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , PhenotypeABSTRACT
Heme oxidation and loss of soluble guanylate cyclase (sGC) is thought to be an important contributor to the development of cardiovascular diseases. Nevertheless, it remains unknown why the heme loses readily in oxidized sGC. In the current study, the conformational change of sGC upon heme oxidation by ODQ was studied based on the fluorescence resonance energy transfer (FRET) between the heme and a fluorophore fluorescein arsenical helix binder (FlAsH-EDT2) labeled at different domains of sGC ß1. This study provides an opportunity to monitor the domain movement of sGC relative to the heme. The results indicated that heme oxidation by ODQ in truncated sCC induced the heme-associated αF helix moving away from the heme, the Per/Arnt/Sim domain (PAS) domain moving closer to the heme, but led the helical domain going further from the heme. We proposed that the synergistic effect of these conformational changes of the discrete region upon heme oxidation forces the heme pocket open, and subsequent heme loss readily. Furthermore, the kinetic studies suggested that the heme oxidation was a fast process and the conformational change was a relatively slow process. The kinetics of heme loss from oxidized sGC was monitored by a new method based on the heme group de-quenching the fluorescence of FlAsH-EDT2.
Subject(s)
Guanylate Cyclase/metabolism , Heme/metabolism , Oxidation-Reduction , Protein Conformation/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Fluorescence Resonance Energy Transfer , Guanylate Cyclase/chemistry , Heme/chemistry , Humans , Kinetics , Nitric Oxide/chemistry , Nitric Oxide/metabolism , Oxadiazoles/pharmacology , Protein Structure, Tertiary/drug effects , Receptors, Cytoplasmic and Nuclear/chemistry , Soluble Guanylyl CyclaseABSTRACT
FBXL5 is a subunit of the SCFFBXL5 ubiquitin ligase complex that targets the proteasomal degradation of iron regulatory protein IRP2, which is an important regulator in iron metabolism. The degradation of FBXL5 itself is regulated in an iron- and oxygen-responsive manner through its diiron center containing Hr-like domain. Although the crystal structure of the Hr-like domain of FBXL5 and its degradation based on iron/oxygen sensing has been reported, the redox sensing molecular mechanism is still not clear. Herein the redox properties of FBXL5 were investigated via EPR, direct electrochemistry, SRCD, fluorescence emission spectroscopy, and redox kinetics. The results indicated that the conformation and function of FBXL5 are tuned by the redox states of the diiron center. The redox reactions of the diiron center are accompanied with conformational changes and iron release, which are associated with FBXL5 stability and degradation. These results provide insights into the redox sensing mechanism by which FBXL5 can serve as an iron metabolism regulator within mammalian cells.
Subject(s)
F-Box Proteins/chemistry , Iron-Regulatory Proteins/chemistry , Oxidation-Reduction , Ubiquitin-Protein Ligase Complexes/chemistry , Circular Dichroism , Crystallography, X-Ray , Electrochemistry , Electron Spin Resonance Spectroscopy , Escherichia coli/metabolism , Humans , Iron/chemistry , Kinetics , Oxygen/chemistry , Protein Domains , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Synchrotrons , Tryptophan/chemistryABSTRACT
Disulfide bond plays crucial roles in stabilization of protein structure and in fine-tuning protein functions. To explore an approach for rational heme protein design, we herein rationally introduced a pair of cysteines (F46C/M55C) into the scaffold of myoglobin (Mb), mimicking those in native neuroglobin. Molecular modeling suggested that it is possible for Cys46 and Cys55 to form an intramolecular disulfide bond, which was confirmed experimentally by ESI-MS analysis, DTNB reaction and CD spectrum. Moreover, it was shown that the spontaneously formed disulfide bond of Cys46-Cys55 fine-tunes not only the heme active site structure, but also the protein functions. The substitution of Phe46 with Ser46 in F46S Mb destabilizes the protein while facilitates H2O2 activation. Remarkably, the formation of an intramolecular disulfide bond of Cys46-Cys55 in F46C/M55C Mb improves the protein stability and regulates the heme site to be more favorable for substrate binding, resulting in enhanced peroxidase activity. This study provides valuable information of structure-function relationship for heme proteins regulated by an intramolecular disulfide bond, and also suggests that construction of such a covalent bond is useful for design of functional heme proteins.
Subject(s)
Disulfides/chemistry , Myoglobin/chemistry , Myoglobin/ultrastructure , Peroxidase/chemistry , Peroxidase/ultrastructure , Protein Engineering/methods , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Computer Simulation , Cysteine/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Structure-Activity RelationshipABSTRACT
Heme proteins perform diverse functions in living systems, of which nitrite reductase (NIR) activity receives much attention recently. In this study, to better understand the structural elements responsible for the NIR activity, we used myoglobin (Mb) as a model heme protein and redesigned the heme active center, by introducing one or two distal histidines, and by creating a channel to the heme center with removal of the native distal His64 gate (His to Ala mutation). UV-Vis kinetic studies, combined with EPR studies, showed that a single distal histidine with a suitable position to the heme iron, i.e., His43, is crucial for nitrite (NO2(-)) to nitric oxide (NO) reduction. Moreover, creation of a water channel to the heme center significantly enhanced the NIR activity compared to the corresponding mutant without the channel. In addition, X-ray crystallographic studies of F43H/H64A Mb and its complexes with NO2(-) or NO revealed a unique hydrogen-bonding network in the heme active center, as well as unique substrate and product binding models, providing valuable structural information for the enhanced NIR activity. These findings enriched our understanding of the structure and NIR activity relationship of heme proteins. The approach of creating a channel in this study is also useful for rational design of other functional heme proteins.
Subject(s)
Heme/chemistry , Myoglobin/chemistry , Nitrite Reductases/chemistry , Animals , Histidine/chemistry , Hydrogen Bonding , Iron/chemistry , Myoglobin/genetics , Nitric Oxide/chemistry , Nitrite Reductases/genetics , Nitrites/chemistry , Protein Engineering , Sperm Whale , Water/chemistryABSTRACT
Heme post-translational modification plays a key role in tuning the structure and function of heme proteins. We herein report a novel tyrosine-heme covalent C−O bond in an artificially produced sperm whale myoglobin (Mb) mutant, F43Y Mb, which formed spontaneously in vivo between the Tyr43 hydroxy group and the heme 4-vinyl group. This highlights the diverse chemistry of heme post-translational modifications, and lays groundwork for further investigation of the structural and functional diversity of covalently-bound heme proteins.