SciCombinator

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Concept: Metalloprotein

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Carbonic anhydrase (CA) is a ubiquitous metalloenzyme with a Zn cofactor coordinated with trigonal histidine imidazole moieties in a tetrahedral geometry. Removal of the Zn cofactor in CA and subsequent binding of Ir afforded CA[Ir]. Under mild and neutral conditions (30 °C, pH 7), CA[Ir] exhibited water-oxidizing activity with a turnover frequency (TOF) of 39.8 min-1, comparable to those of other Ir-based molecular catalysts. Coordination of Ir to the apoprotein of CA is thermodynamically preferred and is associated with an exothermic energy change (ΔH) of -10.8 kcal/mol, implying that the CA apoprotein is stabilized by Ir binding. The catalytic oxygen-evolving activity of CA[Ir] is displayed only when Ir is bound to CA that functions as an effective biological scaffold that activates Ir center for the catalysis. The outcomes of this study indicate that the histidine imidazoles at the CA active site could be exploited as a beneficial biological ligand to provide unforeseen biochemical activity by coordination to a variety of transition metal ions.

Concepts: Carbon dioxide, Enzyme, Hydrogen, Catalysis, Zinc, Transition metal, Enzyme catalysis, Metalloprotein

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The single-molecule properties of metalloproteins have provided an intensely active research area in recent years. This brief review covers some of the techniques used to prepare, measure and analyse the electron transfer properties of metalloproteins, concentrating on scanning tunnelling microscopy-based techniques and advances in attachment of proteins to electrodes.

Concepts: Hemoglobin, Histidine, Single-molecule experiment, Metalloprotein

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Human hemoglobin (HbA) transports molecular oxygen (O2) from the lung to tissues where the partial pressure of O2 is lower. O2 binds to HbA at the heme cofactor and is stabilized by a distal histidine (HisE7). HisE7 has been observed to occupy opened and closed conformations, and is postulated to act as a gate controlling the binding/release of O2. However, it has been suggested that HbA also contains intraprotein oxygen channels for entrances/exits far from the heme. In this study, we developed a novel method of crystal immersion in liquid oxygen prior to X-ray data collection. In the crystals immersed in liquid oxygen, the heme center was oxidized to generate aquomethemoglobin. Increases of structural flexibility were also observed in regions that are synonymous with previously postulated oxygen channels. These regions also correspond to medically relevant mutations which affect O2 affinity. The way HbA utilizes these O2 channels could have a profound impact on understanding the relationship of HbA O2 transport within these disease conditions. Finally, the liquid oxygen immersion technique can be utilized as a new tool to crystallographically examine proteins and protein complexes which utilize O2 for enzyme catalysis or transport.

Concepts: Hemoglobin, Protein, Oxygen, Carbon dioxide, Iron, Chemical bond, Nitrogen, Metalloprotein

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Noncovalent weak interactions [hydrophobic interaction and hydrogen (H)-bond] play crucial roles in controlling the functions of biomolecules, and thus have been used to design artificial metalloproteins/metalloenzymes during the past few decades. In this review, we focus on the recent progresses in protein design by tuning the noncovalent interactions, including hydrophobic and H-bonding interactions. The topics include redesign and reuse of the heme pocket and other protein scaffolds, design of the heme protein interface, and de novo design of metalloproteins. The informations not only give insights into the metalloenzyme reaction mechanisms but also provide new reactions for future applications.

Concepts: Hemoglobin, Protein, Oxygen, Chemical reaction, Creativity, Gauge boson, Protein design, Metalloprotein

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Metalloproteins are essential in biology. The incorporation of metal ion into metalloproteins significantly expands protein functionality and enhances protein stability. Over the last few years, atomic force microscopy-based single molecule force spectroscopy (SMFS) has evolved into a unique tool allowing for probing metalloproteins and metalligand bonds one molecule/bond at a time. Mechanical strength of a wide variety of metalligand bonds has been measured in metal-ligand complexes as well as in metalloproteins, providing detailed information of their underlying free energy profiles and the influence of the protein environment on the bond strength. SMFS experiments have directly demonstrated the effect of the metal binding on the mechanical stability of proteins. Moreover, SMFS has enabled the direct observation of the unfolding and folding of metalloproteins, revealing detailed mechanistic insight into the unfolding pathways modulated by the metal center.

Concepts: Oxygen, Metabolism, Chemistry, Atom, Biochemistry, Force spectroscopy, Metalloprotein, Bioinorganic chemistry

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The binding of nitric oxide (NO) to the heme cofactor of heme-nitric oxide/oxygen binding (H-NOX) proteins can lead to the dissociation of the heme-ligating histidine residue and yield a five-coordinate nitrosyl complex, an important step for NO-dependent signaling. In the five-coordinate nitrosyl complex, NO can reside on either the distal or proximal side of the heme, which could have a profound influence over the lifetime of the in vivo signal. To investigate this central molecular question, we characterized the Shewanella oneidensis H-NOX (So H-NOX)-NO complex biophysically under limiting and excess NO conditions. The results show that So H-NOX preferably forms a distal NO species with both limiting and excess NO. Therefore, signal strength and complex lifetime in vivo will be dictated by the dissociation rate of NO from the distal complex and the rebinding of the histidine ligand to the heme.

Concepts: Hemoglobin, Protein, Amino acid, Nitrogen, Ligand, Heme, Nitric oxide, Metalloprotein

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Thirteen histidines and the α-chain terminal amino group (ACTA) make all of the contributions to the Bohr effect of human hemoglobin. The pKas of the 13 histidines in carbonmonoxy- and deoxyhemoglobin are known from (1)H NMR studies. Those of ACTA are not so precisely known. We employed the Wyman equation and the 13 histidine pairs of pKas to determine the pKas of ACTA by curve-fitting to hemoglobin Bohr effect data. Using all 14 pairs of pKas as preliminary data, we employed the Wyman equation to fit the Bohr data for hemoglobin chemically modified at Cys93β with cystamine, cystine and iodoacetamide. We demonstrate quantitatively that the reduction of the Bohr effect upon chemical modification is due to three negatively contributing Bohr groups: His2β, His77β and His143β. These make twice their normal contributions to the Bohr effect in unmodified hemoglobin. We also find that the ACTA pKas increase with increasing ionic strength.

Concepts: Hemoglobin, Scientific method, Amino acid, Heme, Imidazole, Niels Bohr, Metalloprotein, Bohr effect

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A series of N-substituted saccharins incorporating aryl, alkyl and alkynyl moieties, as well as some ring opened derivatives were prepared and investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The widespread cytosolic isoforms CA I and II were not inhibited by these sulfonamides whereas transmembrane, tumor-associated ones were effectively inhibited, with KIs in the range of 22.1-481nM for CA IX and of 3.9-245nM for hCA XII. Although the inhibition mechanism of these tertiary/secondary sulfonamides is unknown for the moment, the good efficacy and especially selectivity for the inhibition of the tumor-associated over the cytosolic, widespread isoforms, make these derivatives of considerable interest as enzyme inhibitors with various pharmacologic applications.

Concepts: Enzyme, Enzyme inhibitor, Inhibitor, Carbonic anhydrase, Metalloprotein

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Two series of benzenesulfonamide containing isoxazole compounds were prepared by using conventional and microwave (MW) methods. 5-Amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4-carboxamide derivatives were synthesized by the reaction of hydroxymoyl chlorides with 2-cyano-N-(4-sulfamoylphenyl)acetamide in the presence of triethylamine. The synthesized 5-amino isoxazoles were reacted with various benzoyl chlorides in order to obtain 5-amidoisoxazoles. The novel compounds were screened in vitro as inhibitors of four human (h) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1): hCA I, hCA II, hCA IV and hCA VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with KIs in the range of 0.5-49.3nM and hCA VII, a recently validated anti-neuropathic pain target with KIs in the range of 4.3-51.9nM.

Concepts: Carbon dioxide, Enzyme, Synthesis, Novel, Carbonic anhydrase, Protein isoform, Bicarbonate, Metalloprotein

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To increase fidelity of docking/scoring to identify native protein-ligand poses, we have recently introduced the “SQM/COSMO” scoring function which combines semi-empirical quantum mechanical description of noncovalent interactions at the PM6-D3H4X level and COSMO implicit model of solvation. This approach outperformed standard scoring functions but faced challenges with a metalloprotein featuring Zn2+···S- interaction. Here, we invoke SCC-DFTB3-D3H4, higher-level SQM method, and observe an improved behavior for the metalloprotein and retained high-quality results for the other systems. The new method holds promise for diverse protein-ligand complexes including metalloproteins.

Concepts: Scientific method, Function, Interaction, Operator, Identification, Object-oriented programming, Binary relation, Metalloprotein