Noble-metal nanoparticles size and packing density are critical for sensitive surface-enhanced Raman scattering (SERS) and controlled preparation of such films required to achieve reproducibility. ...Provided that they are made reliable, Ag shell on SiO2 microscopic particles (Ag/SiO2) are promising candidates for lab-on-a-bead analytical measurements of low analyte concentration in liquid specimen. Here, we selected nanoporous silica microparticles as a substrate for reduction of AgNO3 with 3-aminopropyltriethoxysilane (APTES). In a single preparation step, homogeneous and continuous films of Ag nanoparticles are formed on SiO2 surfaces with equimolar concentration of APTES and silver nitrate in ethanol. It is discussed that amine and silane moieties in APTES contribute first to an efficient reduction on the silica and second to capping the Ag nanoparticles. The high density and homogeneity of nanoparticle nucleation is further regulated by the nanoporosity of the silica. The Ag/SiO2 microparticles were tested for SERS using self-assembled 4-aminothiophenol monolayers, and an enhancement factor of ca. 2 × 106 is measured. Importantly, the SERS relative standard deviation is 36% when a single microparticle is considered and drops to 11% when sets of 10 microparticles are considered. As prepared, the microparticles are highly suitable for state-of-the-art quantitative lab-on-a-bead interrogation of specimens.
The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O2, NO) requires identification of ligand movement into the binding site and out of the ...enzyme through specific channels and docking sites. We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba 3 from T. thermophilus, which is responsible for the activation and reduction of both O2 and NO, to gain insight into the structure of ligand-binding intermediates at ambient temperature. We show that, upon dissociation, the photolyzed CO becomes trapped within a ligand docking site located near the ring A propionate of heme a 3. The 2131 cm-1 mode of the “docked” CO we have detected corresponds to the B1 state of Mb and persists for 35 μs. The release of CO from the docking site is not followed by recombination to the heme a 3 Fe. Our analysis indicates that this behavior reflects a mechanism in which the protein near ring A of heme a 3 propionate reorganizes about the released CO from the docking site, and establishes a transient barrier that inhibits the recombination process to the heme a 3 Fe for a few milliseconds. Rebinding to heme a 3 occurs with k 2 = 29.5 s-1. These results have implications for understanding the role of ligand binding/escape through docking sites and channels in heme-copper oxidases and, thus, in respiration.
The Thermus thermophilus succinate:quinone reductase (SQR), serving as the respiratory complex II, has been homologously produced under the control of a constitutive promoter and subsequently ...purified. The detailed biochemical characterization of the resulting wild type (wt-rcII) and His-tagged (rcII-His(8)-SdhB and rcII-SdhB-His(6)) complex II variants showed the same properties as the native enzyme with respect to the subunit composition, redox cofactor content and sensitivity to the inhibitors malonate, oxaloacetate, 3-nitropropionic acid and nonyl-4-hydroxyquinoline-N-oxide (NQNO). The position of the His-tag determined whether the enzyme retained its native trimeric conformation or whether it was present in a monomeric form. Only the trimer exhibited positive cooperativity at high temperatures. The EPR signal of the 2Fe-2S cluster was sensitive to the presence of substrate and showed an increased rhombicity in the presence of succinate in the native and in all recombinant forms of the enzyme. The detailed analysis of the shape of this signal as a function of pH, substrate concentration and in the presence of various inhibitors and quinones is presented, leading to a model for the molecular mechanism that underlies the influence of succinate on the rhombicity of the EPR signal of the proximal iron-sulfur cluster.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and ...hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.
Conspectus Cytochrome c oxidase (CcO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven ...CcO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba 3 oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other CcOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba 3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba 3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba 3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba 3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba 3 oxidase.
We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba3andcaa3) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O) under reducing anaerobic ...conditions. The rate of NO consumption and N2O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0± 0.7mol NO/molba3× min and32± 8mol NO/mol caa3× min atNO≈ 50μ M and 20⚬C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme × min), is definitely significant. We also show that for ba3oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar CuB
+coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb3terminal oxidases. Our findings represent functional evidence in support of this hypothesis.
Earlier CO flow-flash experiments on the fully reduced Thermus thermophilus ba3 (Tt ba3) cytochrome oxidase revealed that O2 binding was slowed down by a factor of 10 in the presence of CO (Szundi ...et al., 2010, PNAS 107, 21010–21015). The goal of the current study is to explore whether the long apparent lifetime (∼50 ms) of the CuB+-CO complex generated upon photolysis of the CO-bound mixed-valence Tt ba3 (Koutsoupakis et al., 2019, Acc. Chem. Res. 52, 1380–1390) affects O2 and NO binding and the ability of CuB to act as an electron donor during O-O bond splitting. The CO recombination, NO binding, and the reaction of mixed-valence Tt ba3 with O2 were investigated by time-resolved optical absorption spectroscopy using the CO flow-flash approach and photolabile O2 and NO carriers. No electron backflow was detected after photolysis of the mixed-valence CO-bound Tt ba3. The rate of O2 and NO binding was two times slower than in the fully reduced enzyme in the presence of CO and 20 times slower than in the absence of CO. The purported long-lived CuB+-CO complex did not prevent O-O bond splitting and the resulting PM formation, which was significantly faster (5–10 times) than in the bovine heart enzyme. We propose that O2 binding to heme a3 in Tt ba3 causes CO to dissociate from CuB+ in a concerted manner through steric and/or electronic effects, thus allowing CuB+ to act as an electron donor in the mixed-valence enzyme. The significantly faster O2 binding and O-O bond cleavage in Tt ba3 compared to analogous steps in the aa3 oxidases could reflect evolutionary adaptation of the enzyme to the microaerobic conditions of the T. thermophilus HB8 species.
Drug delivery monitoring and tracking in the human body are two of the biggest challenges in targeted therapy to be addressed by nanomedicine. The ability of imaging drugs and micro-/nanoengineered ...drug carriers and of visualizing their interactions at the cellular interface in a label-free manner is crucial in providing the ability of tracking their cellular pathways and will help understand their biological impact, allowing thus to improve the therapeutic efficacy. We present a fast, label-free technique to achieve high-resolution imaging at the mid-infrared (MIR) spectrum that provides chemical information. Using our custom-made benchtop infrared microscope using a high-repetition-rate pulsed laser (80 MHz, 40 ps), we were able to acquire images with subwavelength resolution (0.8 × λ) at very high speeds. As a proof-of-concept, we embarked on the investigation of nanoengineered polyelectrolyte capsules (NPCs) containing the anticancer drug, docetaxel. These NPCs were synthesized using a layer-by-layer approach built upon a calcium carbonate (CaCO3) core, which was then removed away with ethylenediaminetetraacetic acid. The obtained MIR images show that NPCs are attached to the cell membrane, which is a good step toward an efficient drug delivery. This has been confirmed by both three-dimensional confocal fluorescence and stimulated emission depletion microscopy. Coupled with additional instrumentation and data processing advancements, this setup is capable of video-rate imaging speeds and will be significantly complementing current super-resolution microscopy techniques while providing an unperturbed view into living cells.
PDF
