Fluorescence Quenching of CdTe Nanocrystals by Bound Gold Nanoparticles in Aqueous Solution

Water-soluble gold nanoparticles with an average diameter of 5 nm were prepared with carboxylic acid terminated thiol ligands. These ligands contain zero to eight methylene moieties. CdTe nanocrystals with an average diameter of 5 nm were synthesized with aminoethanethiol capping. These nanocrystals displayed characteristic absorption and emission spectra of quantum dots. The amine terminated CdTe nanocrystals and carboxylic-acid-terminated gold nanoparticles were conjugated in aqueous solution at pH 5.0 by electrostatic interaction, and the conjugation was monitored with fluorescence spectroscopy. The CdTe nanocrystals were significantly quenched upon binding with gold nanoparticles. The quenching efficiency was affected by both the concentration of gold nanoparticles in the complex and the length of spacer between the CdTe nanocrystal and Au nanoparticle. The observed quenching was explained using Förster resonance energy transfer (FRET) mechanism, and the Förster distance was estimated to be 3.8 nm between the donor–acceptor pair.     

The role of the medium in long-range electron transfer

 The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism. Received: 2 January 1997 / Accepted: 6 February 1997     

Sensitive detection of the redox state of copper proteins using fluorescence

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.     

Conformational reorganisation in interfacial protein electron transfer

Protein-protein electron transfer (ET) plays an essential role in all redox chains. Earlier studies which used cross-linking and increased solution viscosity indicated that the rate of many ET reactions is limited (i.e., gated) by conformational reorientations at the surface interface. These results are later supported by structural studies using NMR and molecular modelling. New insights into conformational gating have also come from electrochemical experiments in which proteins are noncovalently adsorbed on the electrode surface. These systems have the advantage that it is relatively easy to vary systematically the driving force and electronic coupling. In this review we summarize the current knowledge obtained from these electrochemical experiments and compare it with some of the results obtained for protein-protein ET.     

Influence of spectral heterogeneity of prodan and laurdan solutions on the transfer of electronic energy to octadecyl rhodamine B

Fluorescence quenching and resonance energy transfer have been studied by steady-state fluorescence spectroscopy. The experimental and theoretical values for the rate constants of the electronic energy transfer (kET) and critical radius (R0) were determined for prodan and laurdan as donors and octadecyl rhodamine B as acceptor. The spectroscopic data show, that prodan and laurdan in solution create an inhomogeneous spectroscopic medium in which multi-channel luminescence phenomena take place. This finding indicated that the modified form of the Stern-Volmer relation should be used for analyzing fluorescence quenching data. Results of performed studies point out, that dipole-dipole interaction is responsible for the resonance energy transfer from prodan and laurdan to octadecyl rhodamine B. The relative quenching efficiencies of both dyes depend on polarity of the medium and are higher for more polar solvent (AcN).     

Monitoring electron transfer by photoacoustic spectroscopy

The electron transfer process between octaethylporphin and quinone molecules dispersed in a polymeric matrix was studied by the photoacoustic technique. It was observed that there was an enhancement of the octaethylporphin photoacoustic signal with an increase of the quinone concentration in the films. This increase appeared to be complementary to octaethylporphin fluorescence quenching and was associated with the electron transfer process. The data were analyzed according to the theory developed by Kaneko for fluorescence data (Kaneko 1992). Correspondence to: R. Sanches     

Photoinduced electron transfer and energy transfer reactions of hydroxo-(2,2′:6′,2″-terpyridine) platinum(II)

The luminescent complex [Pt(terpy)OH]BF₄ undergoes photoinduced electron transfer reactions with phenyl amine electron donors and nitrophenyl electron acceptors. Stern-Volmer analysis of the quenching of metal-to-ligand charge transfer phosphorescence (³MLCT) was used to calculate bimolecular rate constants for electron transfer. Rate constants vary from 10⁸ to >10¹⁰ M⁻¹ s⁻¹, depending on the thermodynamic driving force of the electron transfer reaction, with rate constants indicating that [Pt(terpy)OH]BF₄* is a powerful photo-oxidant. Aromatic triplet energy acceptors can also quench the ³MLCT emission.     

Fluorescence and energy transfer in phycobiliprotein-containing algae at low temperature

Fluorescence emission spectra of Anacystis nidulans, Porphyridium cruentum and Cyanidium caldarium, three phycobiliprotein-containing algae, were measured at temperatures between 4 and 120 K in the absence and in the presence of quinones as quenchers of chlorophyll fluorescence. In all species three major emission bands were observed in the chlorophyll a region, near 685 nm (F-685), 695 nm (F-695) and between 710 and 730 nm. Additional bands were observed at shorter wavelengths; these were preferentially excited by light absorbed by the phycobiliproteins and are presumably due to phycocyanins and allophycocyanins.

The amplitudes of F-685, F-695 and the long-wave emission showed a distinct increase upon cooling. For F-685 and F-695 the temperature dependence was similar to that earlier observed with spinach chloroplasts, for the long-wave emission it appeared to depend on the location of the emission bands, which was different for different species. All three bands were strongly quenched by quinones. These and other data suggest that the origin of these bands is the same as in higher plants, and that the fluorescence increase upon cooling can be explained by a lowering of the efficiency of energy transfer between chlorophyll molecules. It is concluded that at most a small percentage of the emission at 685 nm can be ascribed to allophycocyanin B, and that the efficiency of energy transfer between allophycocyanin B and chlorophyll a probably exceeds 99% both at 77 and 4 K. Experiments with isolated phycobilisomes suggest that energy transfer from allophycocyanin to allophycocyanin B occurs with an efficiency of about 90% at low temperature.

The effect of quenchers can be understood by the assumption that the quenching is caused by the formation of non-fluorescent traps in the bulk chlorophyll. Of three quinones tested, the strongest quenching was observed with dibromothymoquinone, which quenched F-685, F-695 and the long-wave emission approximately equally. Menadione and 1,4-naphthoquinone, however, preferentially quenched the long-wave bands, indicating a stronger interaction with Photosystem I than with Photosystem II chlorophylls.     

DNA-mediated electron transfer

 Electron transfer in DNA has been investigated for decades, but recent experiments highlight our limited fundamental understanding of these processes. Modern electron transfer theory may help to address some of the open mechanistic issues. We summarize and analyze the results of recent experiments from a theoretical perspective. Future research directions are suggested that might help to establish the molecular mechanism(s) for long-range DNA electron transfer. Received, accepted: 5 January 1998     

Chloroplast protein phosphorylation and chlorophyll fluorescence quenching. Activation by tetramethyl-p-hydroquinone, an electron donor to plastoquinone

When tetramethyl-p-benzoquinone (TMQ) is reduced to tetramethyl-p-hydroquinone (TMQH₂) by NaBH₄, TMQH₂ will act as an electron donor in isolated chloroplasts. The resulting electron transport is highly sensitive to inhibition by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the site of donation is inferred to be plastoquinone, in agreement with previous findings. In contrast, when TMQ is added to chloroplasts with ascorbate as reductant, the resulting electron transport is relatively insensitive to DBMIB, and so plastoquinone is assumed not to be involved. In darkness, TMQH₂ activates the chloroplast protein kinase that phosphorylates the light-harvesting chlorophyll a/b-protein complex (LHCP), while TMQ with ascorbate does not. TMQH₂ also activates ATP-dependent chlorophyll fluorescence quenching to a much greater extent than does TMQ with ascorbate. These findings are explained by the recent proposal that phosphorylation of LHCP is activated by reduced plastoquinone. They are therefore evidence for plastoquinone-regulated protein phosphorylation as a mechanism for self-adjustment of distribution of excitation between the two light reactions of photosynthesis.     

Molecular aspects of the electron transfer system which participates in the oxidation of ferrous ion by Thiobacillus ferrooxidans

Abstract: The enzymes and redox proteins, which participate in the oxidation of ferrous ion by the acidophilic iron-oxidizing bacterium Thiobacillus ferrooxidans , have been isolated and characterized. They are Fe(II)-cytochrome c oxidoreductase, cytochromes c -552(s), c -552(m) and c -550(m), rusticyanin, and cytochrome c oxidase. On the basis of the interactions of these components, an electron transfer system has been proposed which seems to function in the oxidation of ferrous ion by the bacterium.     

Ultrafast electron transfer in riboflavin binding protein in macromolecular crowding of nano-sized micelle

In this contribution, we have studied the dynamics of electron transfer (ET) of a flavoprotein to the bound cofactor, an important metabolic process, in a model molecular/macromolecular crowding environments. Vitamin B₂ (riboflavin, Rf) and riboflavin binding protein (RBP) are used as model cofactor and flavoprotein, respectively. An anionic surfactant sodium dodecyl sulfate (SDS) is considered to be model crowding agent. A systematic study on the ET dynamics in various SDS concentration, ranging from below critical micellar concentration (CMC), where the surfactants remain as monomeric form to above CMC, where the surfactants self-assemble to form nanoscopic micelle, explores the dynamics of ET in the model molecular and macromolecular crowding environments. With energy selective excitation in picosecond-resolved studies, we have followed temporal quenching of the tryptophan residue of the protein and Rf in the RBP–Rf complex in various degrees of molecular/macromolecular crowding. The structural integrity of the protein (secondary and tertiary structures) and the vitamin binding capacity of RBP have been investigated using various techniques including UV–Vis, circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) studies in the crowding environments. Our finding suggests that the effect of molecular/macromolecular crowding could have major implication in the intra-protein ET dynamics in cellular environments.     

The absence of energy conservation coupled with electron transfer via the alternative pathway in cyanide-resistant yeast mitochondria

Electron transfer via the alternative pathway in cyanide-resistant mitochondria of the yeast Candida lipolytica is not coupled with ATP synthesis, generation of membrane potential or energy-dependent reverse electron transport in the main respiratory chain. We conclude that during transfer via the alternative pathway no accumulation of energy in the form of high-energy compounds or membrane potential occurs.     

Effects of changes in the capacity for photosynthetic electron transfer and photophosphorylation on the kinetics of fluorescence induction in isolated chloroplasts

Chlorophyll fluorescence, 9-aminoacridine fluorescence and O₂ evolution have been measured in a chloroplast system reconstituted to simulate the induction kinetics observed in leaves. Transients in redox state and energy state, both of which control the yield of fluorescence, were seen to depend upon (a) light intensity, (b) electron-transfer rate as controlled by ferredoxin level, (c) the initial levels of ADP and phosphate and (d) the initial level of NADP. These factors were shown to interact to produce a range of fluorescence patterns. It is suggested that in vivo fluorescence transients in part are due to reduction and phoshorylation of the finite NADP and ADP pools that exist in the chloroplast prior to illumination.     

Effects of protein-protein interactions on electron transfer: docking and electron transfer calculations for complexes between flavodoxin and c-type cytochromes

 Theoretical studies of protein-protein association and electron transfer were performed on the binary systems formed by Desulfovibrio vulgaris Hildenborough (D. v. H.) flavodoxin and D. v. H. cytochrome c ₅₅₃ and by flavodoxin and horse heart cytochrome c. Initial structures for the complexes were obtained by rigid-body docking and were refined by MD to allow for molecular flexibility. The structures thus obtained were analysed in terms of their relative stability through the calculation of excess energies. Electrostatic, van der Waals and solvation energy terms showed all to have significant contributions to the stability of complexes. In the best association solutions found for both cytochromes, these bind to different zones of flavodoxin. The binding site of flavodoxin observed for cytochrome c is in accordance with earlier works [27]. The various association modes found were characterised in terms of electron transfer using the Pathways model. For complexes between flavodoxin and horse heart cytochrome c, some correlation was observed between electron tunnelling coupling factors and conformation energy; the best conformation found for electron transfer corresponded also to the best one in terms of energy. For complexes between flavodoxin and cytochrome c ₅₅₃ this was not the case and a lower correlation was observed between electron tunnelling coupling factors and excess energies. These results are in accordance with the differences in the experimental dependence of electron transfer rates with ionic strength observed between these two cases. Received: 29 December 1998 / Accepted: 22 March 1999     

Amino-terminal amino acid sequences of electron transfer proteins from Gram-negative bacteria as indicators of their cellular localization: the sulfate-reducing bacteria

Abstract Data are presented that all known periplasmic redox proteins from the sulfate reducing bacteria included in the genus, Desulfovibrio have aminoterminal (N-terminal) amino-acid sequences commonly found in other Gram-negative bacteria and are indicative of recognition sites for signal peptides. In contrast, none of the cytoplasmic redox proteins exhibited these unique N-terminal amino-acid sequences. It is proposed that the N-terminal amino-acid residues of a given protein can be used as an indicator of its cellular localization within the bacterial cell.     

Reversible binding of an inorganic cluster-anion to the surface of a gold nanoparticle

Reversible dissociation of POM ligands from electrostatically stabilized monolayer assemblies on metal(0)-nanoparticle surfaces would provide substrates with direct access to reactive metal(0) lattices. As a result, POM-ligand lability could play an important role in catalysis by POM-monolayer protected metal(0) nanoparticles. POM-ligand lability was addressed by first obtained cryogenic transmission electron microscopy (cryo-TEM) images of monolayers of on a 5.4 nm diameter gold-core nanoparticle. These are the smallest nanoparticles on which POM monolayers have been imaged by cryo-TEM. The binding affinities of α-K₉AlW₁₁O₃₉ (K₉1) and α-Na₅AlW₁₂O₄₀ (Na₅2) were obtained from Langmuir isotherms, and the relative labilities of the two ligands were assessed by UV-Vis analysis of changes in surface plasmon resonance (SPR) upon incremental additions of citrate. Thermodynamic constants, K, for monolayer formation by 1 and 2 are 20,000 and 150 M⁻¹, respectively. In timed reactions with citrate, 1 exhibits a substantial kinetic barrier to dissociation, while 2 is rapidly exchanged by the organic protecting ligand.     

Resonance Rayleigh-scattering method for the determination of proteins with gold nanoparticle probe

Gold nanoparticles with a 12-nm diameter were used as probes for the determination of proteins by resonance Rayleigh-scattering techniques. In weak acidic solution, large amounts of citrate anions will self-assemble on the surface of positively charged gold nanoparticles to form supermolecular compounds with negative charges. Below the isoelectric point, proteins with positive charges such as human serum albumin (HSA), bovine serum albumin (BSA), and ovalbumin (Ova) can bind gold nanoparticles to form larger volume products (the diameter of the binding product of gold nanoparticles with HSA is 23 nm.) through electrostatic force, hydrogen bonds, and hydrophobic effects, which can result in a red shift of the maximum absorption wavelength, the remarkable enhancement of the resonance Rayleigh-scattering intensity (RRS), and the appearance of the RRS spectra. At the same time, the second-order-scattering (SOS) and frequency-doubling-scattering (FDS) intensities are also enhanced. The binding products of gold nanoparticles with different proteins have similar spectral characteristics and the maximum wavelengths are located near 303 nm for RRS, 540 nm for SOS, and 390 for FDS, respectively. The scattering enhancement (DeltaI) is directly proportional to the concentration of proteins. Among them, the RRS method has the highest sensitivity and the detection limits are 0.38 ng/ml for HSA, 0.45 ng/ml for BSA, and 0.56 ng/ml for Ova, separately. The methods have good selectivity. A new RRS method for the determination of trace proteins using a gold nanoparticle probe has been developed. Because gold nanoparticle probes do not need to be modified chemically in advance, the method is very simple and fast.     

Direct electron transfer between copper-containing proteins and electrodes

The electrochemistry of some copper-containing proteins and enzymes, viz. azurin, galactose oxidase, tyrosinase (catechol oxidase), and the “blue” multicopper oxidases (ascorbate oxidase, bilirubin oxidase, ceruloplasmin, laccase) is reviewed and discussed in conjunction with their basic biochemical and structural characteristics. It is shown that long-range electron transfer between these enzymes and electrodes can be established, and the mechanistic schemes of the DET processes are proposed.