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An engineered T67C myoglobin mutant undergoes self-oxidation of Cys67 to a sulfinic acid (Cys–SO2H), as confirmed by the X-ray crystal structure, which provides a useful platform to generate artificial proteins by further chemical modifications.

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.

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.

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 (SO

An artificial disulfide bond (Cys46-Cys61) was designed in the heme distal site of myoglobin, which regulates the conformation of the heme distal His64 and the protein reactivity, as confirmed by X-ray crystallography, EPR, and kinetic UV-vis studies. This study shows the successful design of a disulfide bond with suitable positions in globins, conferring a structure and function like those of the native human neuroglobin.

Selenoproteins are a family of special proteins that contain the 21st amino acid, selenocysteine (Sec), in their sequence. Selenoprotein P has 10 Sec residues and modulates selenium homeostasis and redox balance in the brain. Previously, we found that the Sec-devoid His-rich motif of selenoprotein P (Selenop-H) suppressed metal-induced aggregation and neurotoxicities of both Aβ and tau in vitro. To investigate the intervening capacity of Selenop-H on the neuropathology and cognitive deficits of triple transgenic AD (3 × Tg-AD) mice, the Selenop-H gene packaged in rAAV9 was delivered into the hippocampal CA3 regions of mice via stereotaxic injection. Four months later, we demonstrated that Selenop-H (1) improved the spatial learning and memory deficits, (2) alleviated neuron damage and synaptic protein loss, (3) inhibited both tau pathology and amyloid beta protein (Aβ) aggregation, (4) activated both BDNF- and Src-mediated TrkB signaling, and (5) increased MT3 and ZnT3 levels and restored Zn2+ homeostasis in the mice model of AD. The study revealed that Selenop-H is potent in ameliorating AD-related neuropathology and cognitive deficits by modulating TrkB signaling and Zn2+ homeostasis.

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 α

Functional enzyme design has made tremendous progress, but designer enzymes with activities comparable to those of natural enzymes are still limited. In this study, we rationally engineered a funct...

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.

S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) is a unique motif found in several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). Biosynthesis of AviCys requires flavin-dependent Cys decarboxylases, which are highly divergent among different RiPP classes. In this study, we solved the crystal structure of the cypemycin decarboxylase CypD. We show that CypD is structurally highly similar to lanthipeptide decarboxylases despite the absence of sequence similarities between them. We further show that Cys decarboxylases from four RiPP classes have evolved independently and form two major clusters. These results reveal the convergent evolution of AviCys biosynthesis and suggest that all the flavin-dependent Cys decarboxylases likely have a similar Rossmann fold despite their sequence divergences.

The X-ray crystal structure of F43Y/T67R myoglobin revealed unique Tyr-heme double cross-links between Tyr43 and the heme 4-vinyl group, which represents a novel post-translational modification of heme proteins. Moreover, with the feature of a distal His-Arg pair, the designed artificial enzyme exhibited a peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase.

Background: 2',3'-cGAMP (2',3'-cyclic AMP-GMP) has been reported as an agonist of the STING (stimulator of interferon genes) signaling pathway. However, cGAMP has poor membrane permeability and can be hydrolyzed by ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), limiting its ability to activate the STING-IRF3 pathway. This study aimed to investigate that the folate-targeted liposomal cGAMP could overcome the defects of free cGAMP to enhance the antitumor effect. Materials and Methods: cGAMP was encapsulated in PEGylated folic acid-targeted liposomes to construct a carrier-delivered formulation. The particle size and morphology were detected by dynamic light scattering and transmission electron microscopy. The sustained-release ability was measured by drug release and pharmacokinetics. Animal models were applied to evaluate the tumor inhibition efficiency in vivo. Flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction were used to detect the expression of immune cells, secreted cytokines, and target genes. The activation of the STING-IRF3 pathway was evaluated by immunofluorescence. Results: Physical characters of liposomes revealed that the prepared liposomes were stable in neutral humoral environments and released more internal drugs in acidic tumor tissues. Systemic therapy with liposomes on Colorectal 26 tumor-bearing mice in vivo effectively inhibited tumor growth via stimulating the expression of CD8+ T cells and reversed the immunosuppressed tumor microenvironment (TME). Conclusions: The study suggests that the folic acid-targeted cGAMP-loaded liposomes deliver drugs to the TME to enhance the STING agonist activity, improving the efficiency of tumor therapy via the cGAMP-STING-IRF3 pathway.

Inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) have received wide attention for their roles in cancer immunotherapy. It highlights the important role of metalloenzymes in performing human physiological functions. Herein, the recombinant human IDO1 was expressed and purified successfully, and the protein molecule was characterized by SDS-PAGE, MALDI-TOF mass spectrometry, and metalloenzymology. A series of niacin derivatives were investigated with regard to their inhibition on metalloenzyme IDO1, and the resulting potential anti-cancer activities in cell lines. Among the niacin derivatives, 4,4,4-trifluoro-1-(pyridin-3-yl)-butane-1,3-dione (compound 9) was found to be the most effective inhibitor to IDO1 in HepG-2 cells, with an EC50 of 11 µM with low cytotoxicity. The IC50 value of compound 9 with trifluoroethyl group in enzymatic inhibition was shown to be ∼5 times more potent than a positive control 4-phenylimidazole. The interaction between compound 9 and IDO1 was verified by isothermal titration calorimetry and molecular docking study. The most favorable molecular docking results revealed that functional groups of compound 9 contributed to the binding of 9 to IDO1 through IDO1–heme coordination, H-bond interactions and hydrophobic contacts. Our finding provides a strategy for the development of new inhibitor candidates for the therapeutic inhibition of IDO1.

Inhibition of &beta, lactamases presents a promising strategy to restore the &beta, lactams antibacterial activity to resistant bacteria. In this work, we found that aromatic carboxyl substituted 2-triazolylthioacetamides 1a&ndash, j inhibited VIM-2, exhibiting an IC50 value in the range of 20.6&ndash, 58.6 &mu, M. The structure-activity relationship study revealed that replacing the aliphatic carboxylic acid with aromatic carboxyl improved the inhibitory activity of 2-triazolylthioacetamides against VIM-2. 1a&ndash, j (16 mg/mL) restored the antibacterial activity of cefazolin against E. coli cell expressing VIM-2, resulting in a 4&ndash, 8-fold reduction in MICs. The isothermal titration calorimetry (ITC) characterization suggested that the primary binding 2-triazolylthioacetamide (1b, 1c, or 1h) to VIM-2 was a combination of entropy and enthalpy contributions. Further, the crystal structure of VIM-2 in complex with 1b was obtained by co-crystallization with a hanging-drop vapour-diffusion method. The crystal structure analysis revealed that 1b bound to two Zn(II) ions of the enzyme active sites, formed H-bound with Asn233 and structure water molecule, and interacted with the hydrophobic pocket of enzyme activity center utilizing hydrophobic moieties, especially for the phenyl of aromatic carboxyl which formed &pi, &pi, stacking with active residue His263. These studies confirmed that aromatic carboxyl substituted 2-triazolylthioacetamides are the potent VIM-2 inhibitors scaffold and provided help to further optimize 2-triazolylthioacetamides as VIM-2 even or broad-spectrum M&beta, Ls inhibitors.

The design of functional metalloenzymes is attractive, but few designs can exceed the catalytic rate of native enzymes. In order to create an effective artificial dehaloperoxidase (DHP), we redesigned the heme center of myoglobin (Mb) by introducing a distal Tyr43 and replacing the distal His64 with an Asp, which combines the structural features of chloroperoxidase (a distal Asp) with DHP (a distal Tyr). The rationally designed F43Y/H64D Mb was shown to exhibit a remarkable dehalogenation activity, with a catalytic efficiency more than 1000-fold higher than that of a native DHP from A. ornata. To the best of our knowledge, this is the highest activity reported to date for an artificial DHP. Moreover, X-ray structures of F43Y/H64D Mb and in complex with a typical substrate, 2,4,6-trichlorophenol (TCP), revealed a distal substrate binding site and crucial roles of both distal Tyr43 and Asp64. This study not only provides insights into the structure and function relationship of native and artificial DHPs but...

The structure and function of heme proteins are regulated by diverse post-translational modifications including heme-protein cross-links, with the underlying mechanisms not well understood. In this study, we introduced a Cys (K42C) close to the heme 4-vinyl group in sperm whale myoglobin (Mb) and solved its X-ray crystal structure. Interestingly, we found that K42C Mb can partially form a Cys-heme cross-link (termed K42C Mb-X) under dithiothreitol-induced reductive conditions in presence of O 2 , as suggested by guanidine hydrochloride-induced unfolding and heme extraction studies. Mass spectrometry (MS) studies, together with trypsin digestion studies, further indicated that a thioether bond is formed between Cys42 and the heme 4-vinyl group with an additional mass of 16 Da, likely due to hydroxylation of the α‑carbon. We then proposed a plausible mechanism for the formation of the novel Cys-heme cross-link based on MS, kinetic UV–vis and electron paramagnetic resonance (EPR) studies. Moreover, the Cys-heme cross-link was shown to fine-tune the protein reactivity toward activation of H 2 O 2 . This study provides valuable insights into the post-translational modification of heme proteins, and also suggests that the Cys-heme cross-link can be induced to form in vitro , making it useful for design of new heme proteins with a non-dissociable heme and improved functions.

Alzheimer’s disease (AD) is one of the leading causes of death for people over 65 years. Worse still, no completely effective therapeutic agent is available so far. One important pathological hallmark of AD is accumulated amyloid-β (Aβ) plaques with dysregulated metal homeostasis. Human metallothionin 3 (MT3), a regulator of metal homeostasis, is downregulated at least 30% in AD brain. So far, some in vitro studies demonstrated its multiple functions related to AD. However, it is a great pity that systematic in vivo studies of MT3 on AD model animals are still a blank so far. In this study, we treated APP/PS1 mice with sustained drug release of Zn7MT3 directly to the central nervous system, and investigated the role and molecular mechanism of Zn7MT3 to protect against AD mice systematically. The results demonstrated that Zn7MT3 can significantly ameliorate cognitive deficits, regulate metal homeostasis, abolish Aβ plaque load, and reduce oxidative stress. Additionally, it has been confirmed that MT3 is penetrable to the blood brain barrier of AD mice. All these results support that Zn7MT3 is an effective AD suppressing agent and has potential for applications in Alzheimer’s disease therapy.

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.

To reveal the structure-function relationship of heme proteins, and to provide clues for creating artificial heme proteins with improved functions, we here use myoglobin (Mb) as a model protein, and report that its peroxidase activity can be enhanced by construction of two distal histidines and a channel to the heme pocket. It showed that in addition to a single distal histidine with a suitable distance to the heme iron (Phe43 to His43 mutation), a second distal histidine (Leu29 to His29 mutation) can work cooperatively to increase the turnover number, mimicking the role of well-known His-Arg pair in native peroxidases. Moreover, a channel created to the heme pocket by removal of the native His64 gate (His64 to Ala64 mutation) was shown to facilitate the binding of substrate, resulting in enhanced catalytic efficiency for the triple mutant L29H/F43H/H64A Mb, which is beyond the addition of both double mutants, L29H/H64A Mb and F43H/H64A Mb. These results provide valuable information for elucidating the structure-function relationship of heme proteins. In addition, this study provides clues for design of artificial heme proteins, and the strategy of creating a channel to the heme active center is expected be extended to design of other artificial enzymes with improved catalytic performance.

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.

A de novo designed intramolecular disulfide bond in myoglobin, resembling that in cytoglobin without structural evidence, was confirmed by an X-ray structure for the first time and was demonstrated to regulate both the structure and function of this protein, which fulfills the design of an artificial dehaloperoxidase, with an activity exceeding that of a native enzyme.

Although heme proteins are extensively studied, the structure and hydrolysis activity relationship of heme proteins is still not well understood. We herein made a comparison of the heme active site of myoglobin (Mb) and its two distal mutants, L29H Mb and L29E Mb, and found that the heme distal hydrogen-bonding network regulates the hydrolysis activity, with L29E Mb being more reactive. Fluoride competitive binding studies revealed that L29E Mb has an axial water molecule readily replaced by fluoride anion, whereas L29H Mb has a much more stable axial water molecule. pH titration studies showed that L29E Mb exhibits a lower p K a and L29H Mb exhibits a higher p K a of the heme coordinated water molecule compared to that of Mb, rationalizing the relative hydrolysis activities. Moreover, pH-jump induced unfolding studies suggested that the heme distal hydrogen-bonding network, especially in L29H Mb, plays a key role in stabilization of the protein. This study thus provides valuable information for understanding the structure and hydrolysis activity relationship of heme proteins, as well as for rational protein design with the knowledge of structural and functional diversity of heme proteins.

A heme-protein cross-link is a key post-translational modification (PTM) of heme proteins. Meanwhile, the structural and functional consequences of heme-protein cross-links are not fully understood, due to limited studies on a direct comparison of the same protein with and without the cross-link. A Tyr-heme cross-link with a C-O bond is a newly discovered PTM of heme proteins, and is spontaneously formed in F43Y myoglobin (Mb) between the Tyr hydroxyl group and the heme 4-vinyl group in vivo. In this study, we found that with an additional distal His29 introduced in the heme pocket, the double mutant L29H/F43Y Mb can form two distinct forms under different protein purification conditions, with and without a novel Tyr-heme cross-link. By solving the X-ray structures of both forms of L29H/F43Y Mb and performing spectroscopic studies, we made a direct structural and functional comparison in the same protein scaffold. It revealed that the Tyr-heme cross-link regulates the heme distal hydrogen-bonding network, and fine-tunes not only the spectroscopic and ligand binding properties, but also the protein reactivity. Moreover, the formation of the Tyr-heme cross-link in the double mutant L29H/F43Y Mb was investigated in vitro. This study addressed the key issue of how Tyr-heme cross-link fine-tunes the structure and functions of the heme protein, and provided a plausible mechanism for the formation of the newly discovered Tyr-heme cross-link.

Heme coordination state determines the functional diversity of heme proteins. Using myoglobin as a model protein, we designed a distal hydrogen-bonding network by introducing both distal glutamic acid (Glu29) and histidine (His43) residues and regulated the heme into a bis-His coordination state with native ligands His64 and His93. This resembles the heme site in natural bis-His coordinated heme proteins such as cytoglobin and neuroglobin. A single mutation of L29E or F43H was found to form a distinct hydrogen-bonding network involving distal water molecules, instead of the bis-His heme coordination, which highlights the importance of the combination of multiple hydrogen-bonding interactions to regulate the heme coordination state. Kinetic studies further revealed that direct coordination of distal His64 to the heme iron negatively regulates fluoride binding and hydrogen peroxide activation by competing with the exogenous ligands. The new approach developed in this study can be generally applicable for fine-tuning the structure and function of heme proteins.

The rational design of metalloenzymes provides advantages not only for illustrating the structure and function relationship of native enzymes, but also for creating functional artificial enzymes comparable to native enzymes. Dye-decolorizing peroxidases (DyPs) are a new family of heme peroxidases and have received much attention recently. Inspired by the structural features of native DyPs with multiple Tyr and Trp residues, we herein aimed to design functional artificial DyPs using myoglobin (Mb), an O2 carrier as a protein scaffold, by further introduction of Tyr/Trp into the secondary sphere of the heme center in the F43Y Mb mutant. The latter has been shown to possess a novel Tyr–heme cross-link and exhibit enhanced peroxidase activity, which provides an ideal platform to design a series of derivatives, including F43Y/F46Y Mb, F43Y/I107Y Mb, F43Y/F138 W Mb and F43Y/I107Y/F138 W Mb. Our design revealed that the Tyr–heme cross-link was well-retained in the mutants except for F43Y/F46Y Mb, as confirmed by X-ray crystal structure analysis. More importantly, stopped-flow kinetic studies showed that these derivatives exhibit enhanced dye-decolorizing peroxidase activities compared to that of wild-type (WT) Mb. This is particularly the case for the double mutant F43Y/F138 W Mb, exhibiting an overall catalytic efficiency (kcat/Km) of 110 670 M−1 s−1, which is ∼144-fold and ∼20-fold that of WT Mb and F43Y Mb, respectively, and is ∼4.3-fold that of native DyP from Vibrio cholerae. Stopped-flow, electron paramagnetic resonance (EPR) and isothermal titration calorimetry (ITC) studies further provided insights into the activation of H2O2 and the binding of a substrate, reactive blue 19 (RB19), to the double mutant. This study provides valuable information for elucidating the structure and dye-decolorizing function relationship of peroxidases, and also clues for the design of other functional artificial heme enzymes.

Iron-containing enzymes such as heme enzymes play crucial roles in biological systems. Three distinct heme-containing dioxygenase enzymes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 1 (IDO1) and indoleamine 2,3-dioxygenase 2 (IDO2) catalyze the initial and rate-limiting step of L-tryptophan catabolism through the kynurenine pathway in mammals. Overexpression of these enzymes causes depletion of tryptophan and the accumulation of metabolic products, which contributes to tumor immune tolerance and immune dysregulation in a variety of disease pathologies. In the past few decades, IDO1 has garnered the most attention as a therapeutic target with great potential in cancer immunotherapy. Many potential inhibitors of IDO1 have been designed, synthesized and evaluated, among which indoximod (D-1-MT), INCB024360, GDC-0919 (formerly NLG-919), and an IDO1 peptide-based vaccine have advanced to the clinical trial stage. However, recently, the roles of TDO and IDO2 have been elucidated in immune suppression. In this review, the current drug discovery landscape for targeting TDO, IDO1 and IDO2 is highlighted, with particular attention to the recent use of drugs in clinical trials. Moreover, the crystal structures of these enzymes, in complex with inhibitors, and the mechanisms of Trp catabolism in the first step, are summarized to provide information for facilitating the discovery of new enzyme inhibitors.

Alzheimer’s disease (AD) is one of the leading causes of death for people over 65 years. Worse still, no completely effective therapeutic agent is available so far. One important pathological hallmark of AD is accumulated amyloid-β (Aβ) plaques with dysregulated metal homeostasis. Human metallothionin 3 (MT3), a regulator of metal homeostasis, is downregulated at least 30% in AD brain. So far, some in vitro studies demonstrated its multiple functions related to AD. However, it is a great pity that systematic in vivo studies of MT3 on AD model animals are still a blank so far. In this study, we treated APP/PS1 mice with sustained drug release of Zn7MT3 directly to the central nervous system, and investigated the role and molecular mechanism of Zn7MT3 to protect against AD mice systematically. The results demonstrated that Zn7MT3 can significantly ameliorate cognitive deficits, regulate metal homeostasis, abolish Aβ plaque load, and reduce oxidative stress. Additionally, it has been confirmed that MT3 is penetrable to the blood brain barrier of AD mice. All these results support that Zn7MT3 is an effective AD suppressing agent and has potential for applications in Alzheimer’s disease therapy.

Soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. NO binds to the heme of sGC to catalyze the synthesis of the second messenger cGMP, which plays a critical role in several physiological processes. However, the molecular mechanism for sGC to mediate the NO signaling remains unclear. Here fluorophore FlAsH-EDT2 and fluorescent proteins were employed to study the NO-induced sGC activation. FlAsH-EDT2 labeling study revealed that NO binding to the H-NOX domain of sGC increased the distance between H-NOX and PAS domain and the separation between H-NOX and coiled-coil domain. The heme pocket conformation changed from “closed” to “open” upon NO binding. In addition, the NO-induced conformational change of sGC was firstly investigated in vivo through fluorescence lifetime imaging microscopy. The results both in vitro and in vivo indicated the conformational change of the catalytic domain of sGC from “open” to “closed” upon NO binding. NO binding to the heme of H-NOX domain caused breaking of Fe-N coordination bond, initiated the domain moving and conformational change, induced the allosteric effect of sGC to trigger the NO-signaling from H-NOX via PAS & coiled-coil to the catalytic domain, and ultimately stimulates the cyclase activity of sGC.

In this work, direct electrochemistry and electrocatalytic activity of four new kinds of artificially prepared superoxide dismutases (SODs) from human pathogen Clostridium difficile with different metal centers and microenvironments, that is, manganese superoxide dismutase (Mn-SOD), azide ion-coordinated manganese superoxide dismutase (Mn(N 3 − )-SOD), iron superoxide dismutase (Fe-SOD), and nickel superoxide dismutase (Ni-SOD), are systematically investigated. The direct electron transfer of the SODs is realized by 3-mercaptopropionic acid (MPA) confined on gold flowers electrodeposited onto glassy carbon electrode. The electrochemical parameters are dependent on the metal centers and microenvironments of the SODs with respect to the formal potential, reversibility of electrode reactions, and kinetic rate constant. Furthermore, except Ni-SOD, the other three kinds of SODs all exhibit a bifunctional electrocatalytic activity toward O 2 − . The combination of the enhanced electron transfer and the enzyme catalytic activities of SODs enables a sensitive and selective determination of O 2 − .

Soluble guanylatecyclase (sGC), as a NO receptor, is a key metalloenzyme in mediating NO-signaling transduction. sGC is activated by NO to catalyze the conversion of guanosine 5-triphosphate (GTP) to cyclic guanylate monophosphate (cGMP). The dysfunction of NO signaling results in many pathological disorders, including several cardiovascular diseases such as arterial hypertension, pulmonary hypertension, heart failure and so on. Significant advances in its structure, function, mechanism, and physiological & pathological roles have been made throughout the past fifteen years. We herein review the progress of sGC on structural, functional investigations, as well as the proposed activation/deactivationmechanism. The heme-dependent sGC stimulators and heme-independent sGC activators have also been summarized briefly.

Nitric oxide (NO), a signaling molecule in the cardiovascular system, has been receiving increasing attention since Furchgott, Ignarro, and Murad were awarded the Nobel Prize in Physiology and Medicine for the discovery in 1998. Soluble guanylate cyclase (sGC), as an NO receptor, is a key metalloprotein in mediating NO signaling transduction. sGC is activated by NO to catalyze the conversion of guanosine 5′-triphosphate (GTP) to cyclic guanylate monophosphate (cGMP). The dysfunction of NO signaling results in many pathological disorders, including several cardiovascular diseases, such as arterial hypertension, pulmonary hypertension, heart failure and so on. Significant advances in its structure, function, mechanism, and physiological and pathological roles have been made throughout the past 15 years. We herein review the progress of sGC on structural, functional investigations, as well as the proposed activation/deactivation mechanism. The heme-dependent sGC stimulators and heme-independent sGC activators have also been summarized briefly.

Aggregation and cytotoxicity of Aβ with redox-active metals in neuronal cells have been implicated in the progression of Alzheimer disease. Human metallothionein (MT) 3 is highly expressed in the normal human brain and is downregulated in Alzheimer disease. Zn(7)MT3 can protect against the neuronal toxicity of Aβ by preventing copper-mediated Aβ aggregation, abolishing the production of reactive oxygen species (ROS) and the related cellular toxicity. In this study, we intended to decipher the roles of single-domain proteins (α/β) and the α-β domain-domain interaction of Zn(7)MT3 to determine the molecular mechanism for protection against the neuronal cytotoxicity of Aβ(1-42) with copper ions. With this in mind, the α and β single-domain proteins, heterozygous β(MT3)-α(MT1), and a linker-truncated mutant ∆31-34 were prepared and characterized. In the presence/absence of various Zn(7)MT3 proteins, the Aβ(1-42)-Cu(2+)-mediated aggregation, the production of ROS, and the cellular toxicity were investigated by transmission electron microscopy, ROS assay by means of a fluorescent probe, and SH-SY5Y cell viability, respectively. The β domain cannot abolish Aβ(1-42)-Cu(2+)-induced aggregation, and neither the β domain nor the α domain can quench the production of ROS because of the redox cycling of Aβ-Cu(2+). Similarly to wild-type Zn(7)MT3, the heterozygous β(MT3)-α(MT1) possesses the characteristic of alleviating Aβ(1-42) aggregation and oxidative stress to neuronal cells. Therefore, the two domains through the linker Lys-Lys-Ser form a cooperative unit, and each of them is indispensable in conducting its bioactivity. The α domain plays an important role in modulating the stability of the metal-thiolate cluster, and the α-β domain-domain interaction through the linker is critical for its protective role in the brain.

A hydrogen-bond (H-bond) network, specifically a Tyr-associated H-bond network, plays key roles in regulating the structure and function of proteins, as exemplified by abundant heme proteins in nature. To explore an approach for fine-tuning the structure and function of artificial heme proteins, we herein used myoglobin (Mb) as a model protein and introduced a Tyr residue in the secondary sphere of the heme active site at two different positions (107 and 138). We performed X-ray crystallography, UV-Vis spectroscopy, stopped-flow kinetics, and electron paramagnetic resonance (EPR) studies for the two single mutants, I107Y Mb and F138Y Mb, and compared to that of wild-type Mb under the same conditions. The results showed that both Tyr107 and Tyr138 form a distinct H-bond network involving water molecules and neighboring residues, which fine-tunes ligand binding to the heme iron and enhances the protein stability, respectively. Moreover, the Tyr107-associated H-bond network was shown to fine-tune both H2O2 binding and activation. With two cases demonstrated for Mb, this study suggests that the Tyr-associated H-bond network has distinct roles in regulating the protein structure, properties and functions, depending on its location in the protein scaffold. Therefore, it is possible to design a Tyr-associated H-bond network in general to create other artificial heme proteins with improved properties and functions.

The interaction between chloramphenicol (CHL) and neuroglobin (Ngb) has been investigated by using fluorescence, synchronous fluorescence, UV-Vis and circular dichroism (CD) spectroscopy. It has been found that CHL molecule can quench the intrinsic fluorescence of Ngb in a way of dynamic quenching mechanism, which was supported by UV-Vis spectral data. Their effective quenching constants (KSV) are2.2×104,2.6×104,and 3.1×104 L⋅mol−1at 298 K, 303 K, and 308 K, respectively. The enthalpy change (ΔH) and entropy change (ΔS) for this reaction are 26.42 kJ⋅mol−1and 171.7 J⋅K−1, respectively. It means that the hydrophobic interaction is the main intermolecular force of the interaction between CHL and Ngb. Synchronous fluorescence spectra showed that the microenvironment of tryptophan and tyrosine residues of Ngb has been changed slightly. The fluorescence quenching efficiency of CHL to tyrosine residues is a little bit more than that to tryptophan residues of Ngb. Furthermore, CD spectra indicated that CHL can induce the formation of α-helix of Ngb.