Electrical fields in membrane proteins]

The electric fields created by dipoles of the peptide bonds of alpha-helices of membrane proteins are considered. It has been shown that the electric field of the alpha-helix compensates for the loss of the Born hydration energy and promotes dissociation of the carboxyl groups located at the depth of up to 5 A from the water surface. The presence of the carboxylate anion facilitates penetration of the hydronium ion into the membrane and lowers the potential barrier by 0.1-0.2 eV (depending on the membrane thickness). A three-layer model of the reaction centre of photosynthetic bacteria is proposed. An estimate of the dielectric constant of different parts of the reaction centre is obtained by means of comparison of photoinduced electrogenetic transmembrane potential displacement with structural data. Estimates of the electric potentials at the electron transfer chain cofactors induced by the alpha-helical segments of the reaction centre protein are given. It is shown that the asymmetry in the location of alpha-helices affects significantly the redox potentials of the electron carriers and lead to a kinetic advantage of the A-chain of electron transfer over the B-chain.     

Artificial peptides with unnatural components designed for materializing protein function

In order to design functional peptides, we employed two strategies. The first one is to incorporate rigid unnatural amino acids into peptides to make the peptide backbone rigid. Functions were expected to appear through the conformational control by the strategy. A series of cyclic peptides constituted of alternating natural amino acids and 3-aminobenzoic acid, used as an unnatural amino acid, were synthesized. These cyclic peptides were found to function as strong binders for phosphomonoester, catalysts for ester hydrolysis, and/or ion channels. The second strategy is to conjugate peptides with unnatural and inherently functional molecules. Following this strategy, oligo(L-leucine)- or oligo(L-phenylalanine)-modified ruthenium tris(bipyridine) complexes were synthesized. Distance dependence of the photoinduced electron transfer from the ruthenium complexes and the function as sensors for phosphate anion (H(2)PO(-)(4)) are discussed.     

Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity quinone binding site (Q(H)), using N-methylnicotinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase occurred in two phases as judged from kinetic difference spectra. In the faster phase, the transient species with an absorption maximum at 440 nm, a characteristic of the formation of ubisemiquinone anion radical, appeared within 10 micros after pulse radiolysis. In the slower phase, a decrease of absorption at 440 nm was accompanied by an increase of absorption at 428 and 561 nm, characteristic of the reduced form. In contrast, with the bound ubiquinone-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, though the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at the Q(H) site to heme b exists in cytochrome bo. The first-order rate constant of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecular electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W. J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908], indicating that electron transfer from the bound ubisemiquinone at the Q(H) site to the hemes slows down at the alkaline pH where the bound ubisemiquinone can be stabilized. These findings are consistent with our previous proposal that the bound ubiquinone at the Q(H) site mediates electron transfer from the low-affinity quinol oxidation site in subunit II to low-spin heme b in subunit I.     

Impedance spectroscopy as a tool for non‐intrusive detection of extracellular mediators in microbial fuel cells

Endogenously produced, diffusible redox mediators can act as electron shuttles for bacterial respiration. Accordingly, the mediators also serve a critical role in microbial fuel cells (MFCs), as they assist extracellular electron transfer from the bacteria to the anode serving as the intermediate electron sink. Electrochemical impedance spectroscopy (EIS) may be a valuable tool for evaluating the role of mediators in an operating MFC. EIS offers distinct advantages over some conventional analytical methods for the investigation of MFC systems because EIS can elucidate the electrochemical properties of various charge transfer processes in the bio‐energetic pathway. Preliminary investigations of Shewanella oneidensis DSP10‐based MFCs revealved that even low quantities of extracellular mediators significantly influence the impedance behavior of MFCs. EIS results also suggested that for the model MFC studied, electron transfer from the mediator to the anode may be up to 15 times faster than the electron transfer from bacteria to the mediator. When a simple carbonate membrane separated the anode and cathode chambers, the extracellular mediators were also detected at the cathode, indicating diffusion from the anode under open circuit conditions. The findings demonstrated that EIS can be used as a tool to indicate presence of extracellular redox mediators produced by microorganisms and their participation in extracellular electron shuttling. Biotechnol. Bioeng. 2009; 104: 882–891. © 2009 Wiley Periodicals, Inc.     

The reaction between diethylpyrocarbonate and sulfhydryl groups in carbocylate buffers.

Diethylpyrocarbonate reacts with sulfhydryl groups in the presence of carboxylate buffers to form a product which absorbs at 242 nm. The product is believed to be a thiol ester formed from the sulfhydryl compound and the buffer anion. This reaction interferes with the use of diethylpyrocarbonate to determine protein histidine residues when the reaction is performed in carboxylate buffers.     

Two Channels of Charge Generation in Perylene Monoimide Solid‐State Dye‐Sensitized Solar Cells

The mechanism of charge generation in solid‐state dye‐sensitized solar cells using triarylamine‐substituted perylene monoimide dyes is studied by vis‐NIR broadband pump‐probe transient absorption spectroscopy. The experiments demonstrate that photoinduced electron injection into the TiO₂ can only occur in regions where Li⁺, from the commonly used Li‐TFSI additive salt, is present on the TiO₂ surface. Incomplete surface coverage by Li⁺ means that some dye excitons cannot inject their electron into the TiO₂. However it is observed in the solar cell structure that some of the dye excitons that cannot directly inject an electron still contribute to free charge generation by the previously hypothesized reductive quenching mechanism (hole transfer to the solid‐state hole transporter followed by electron injection from the dye anion into the TiO₂). The contribution of reductive quenching to the quantum efficiency of charge generation is significant, raising it from 68% to over 80%. Optimization of this reductive quenching pathway could be exploited to maintain high quantum efficiency in dyes with greater NIR absorption to achieve overall enhancements in device performance. It is demonstrated that broadband NIR transient spectroscopy is necessary to obtain population kinetics in these systems, as strong Stark effects distort the population kinetics in the visible region.     

Simple PbII fluorescent probe based on PbII-catalyzed hydrolysis of phosphodiester

A new fluorescent probe for PbII, p-nitrophenyl 3H-phenoxazin-3-one-7-yl phosphoric acid (NPPA), was designed and synthesized by linking resorufin (serving as a fluorophore and electron acceptor) to p-nitrophenol (serving as a fluorescence quencher and electron donor) through phosphodiester bonds. When NPPA was irradiated with light, intramolecular fluorescence self-quenching took place because of the photoinduced electron transfer from the donor to the acceptor. However, upon the addition of PbII, the phosphate ester bonds in the probe were cleaved and the fluorophore was released, accompanying the retrievement of fluorescence.     

Detecting photoinduced electron transfer processes in the SERS spectrum of salicylate anion adsorbed on silver nanoparticles

The surface-enhanced Raman scattering (SERS) of salicylic acid (S) adsorbed on a silver sol in H(2)O and D(2)O has been investigated. At pH 5 or greater, the adsorbed species is the salicylate anion (2-hydroxybenzoate anion) (S(-)), which links to the metal nanoparticle (Ag(n)) through the carboxylate group (S(-)-Ag(n)). We demonstrate that the selective enhancement of the bands is due mainly to a resonant electron or charge transfer process (ET or CT) from the metallic nanoparticle to the adsorbate, yielding the transient formation of the respective radical dianion (S.(2-)-Ag(n) (+)). It is found that the enhanced bands, and especially mode 8a;nu(ring), are related to the differences between the equilibrium structures of the adsorbate in its ground (S(-)) and CT-excited states (S.(2-)).     

Interaction of cartilage proteoglycans with hyaluronic acid. The role of the hyaluronic acid carboxyl groups.

Hyaluronic acid-derived oligomers of five to fifteen repeat dissaccharides effectively bind to bovine nasal-cartilage proteoglycan and inhibit the interaction between proteoglycans and high-molecular-weight hyaluronic acid. If, however, the hyaluronic acid oligosaccharides are modified by reaction with diazomethane to form the carboxyl methyl esters of the glucuronic acid residues, their inhibitory activity is abolished. The binding capacity can be fully restored by saponification. The amide derivative, which is formed by condensation of the oligosaccharide carboxyl groups with glycine methyl ester, is also ineffective in blocking the proteoglycan-hyaluronic acid interaction. In this case, binding activity is not restored when the amidated oligomers are subjected to saponification to yield the free carboxylate groups on the glycine residues. Thus the displacement of the carboxylate groups on the polysaccharide chain by the interposition of a glycine residue blocks the interaction between the proteoglycans and the hyaluronic acid oligomers. When the oligosaccharide methyl ester is reduced with NaBH4, the resultant glucose-containing oligomers exhibit decreased binding to proteoglycans. Thus it appears that the hyaluronic acid carboxylate anion in a specific spatial orientation is required for hyaluronic acid-proteoglycan interaction.     

Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

The one-electron reduction of uroporphyrin I by rat hepatic microsomes

Uroporphyrin I, which accumulates in body tissues of congenital erythropoietic porphyria patients, can undergo an enzymatic one-electron reduction to the porphyrin anion radical when a suitable reducing cofactor is present. We have demonstrated, in the absence of light, that anaerobic microsomal incubations containing NADPH and uroporphyrin I give an electron spin resonance spectrum consistent with the enzymatic formation of a porphyrin anion free radical. This radical undergoes a second-order decay (k2 approximately 10(5) M-1 s-1) due to nonenzymatic disproportionation of the radical. Aerobic microsomal incubations were also investigated for the reduction of oxygen to superoxide by monitoring oxygen consumption and the spin-trapping of superoxide. These experiments demonstrate that electron transfer from the porphyrin radical to molecular oxygen does occur, but due to the slow formation of the radical anion, no oxygen consumption above the basal level could be detected in the microsomal incubations. The photoreduction of uroporphyrin I in aerobic and anaerobic incubations was also investigated.     

Catalysis of electron transfer across phospholipid bilayers by iron-porphyrin complexes

Phospholipid vesicles containing K₃Fe(CN)₆ were prepared from egg yolk phosphatidylcholine. Hemin dimethyl ester was incorporated into these vesicles during preparation in ratios of phospholipid to hemin dimethyl ester that varied from 200 : 1 to 45000 : 1. Electron transfer across the bilayer was measured anaerobically after injecting the vesicles into a solution containing reduced indigotetrasulfonic acid. Vesicles containing hemin dimethyl ester exhibited high rates of electron transfer (240 electrons/molecule hemin dimethyl ester per min). Conditions could be selected where the rate-limiting step for catalysis was either the bimolecular reaction between ferric hemin dimethyl ester and reduced indigotetrasulfonic acid or the movement of hemin dimethyl ester from interface to interface. The hemin dimethyl ester-catalyzed electron transfer went to completion within a few seconds, completely oxidizing the reduced indigotetrasulfonic acid. Valinomycin (in the presence of potassium) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone were without effect on catalyzed electron transport. Thus, the electron transport is not electrogenic but is a coupled, neutral system. By specific assay, neither phosphate nor cyanide was significantly transported during electron transfer but evidence is provided to suggest that a coordinated hydroxide accompanies movement of Fe(III) hemin dimethyl ester from the inside surface to the outside surface of the bilayer. It was also demonstrated in a bulk phase transport system that hemin dimethyl ester readily catalyzes transfer of S¹⁴CN^? through a chloroform layer separating two aqueous phases. Another more hydrophobic iron-porphyrin complex, Fe(III) tetraphenylporphyrin, was found to be twice as effective as hemin dimethyl ester. Other porphyrin complexes were also tested as control systems. No significant catalysis was found for metal-free protoporphyrin IX dimethyl ester or Ni(II) tetraphenylporphyrin. The results are discussed in comparison with in vivo electron transport and the future usefulness of this model system.     

Poly(m-phenylenediamine) nanospheres and nanorods: selective synthesis and their application for multiplex nucleic acid detection

In this paper, we demonstrate for the first time that poly(m-phenylenediamine) (PMPD) nanospheres and nanorods can be selectively synthesized via chemical oxidation polymerization of m-phenylenediamine (MPD) monomers using ammonium persulfate (APS) as an oxidant at room temperature. It suggests that the pH value plays a critical role in controlling the the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can be used as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum system is also demonstrated.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T₀ mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Photoacoustic diagnostics of fast photochemical and photobiological processes. Analysis of inverse problem solution

Photoacoustic (PA) diagnostics is a time-resolved experimental technique that measures transient photoinduced volume changes on the nano- and microsecond time-scales. The technique has been used to study the energetics and dynamics of chemical and biochemical reactions initiated by absorption of light. The dynamics of the volume changes and associated relaxation processes can be restored from PA-waveforms by solving an ill-posed problem of deconvolution. For the systems with known relaxation kinetics scheme this problem is usually solved by an iterative approximation algorithm. In complex photoactive systems (e.g. photosynthetic samples), where information about kinetics of fast photoinduced volume changes is not available, an algorithm of direct deconvolution must be used. The implementation of one of the linear deconvolution algorithms (Tikhonov's alpha-regularization) for the PA-diagnostics is therefore considered. The problem of the optimal choice of experimental set-up, and restoration algorithm is analyzed by a numerical simulation. It is shown that the quality of PA-diagnostic experiment is mainly determined by a transfer function converting the relaxation spectrum to the spectrum of output electric signal. The analytical expressions to calculate this function (so called PA-filter) are presented. The performance of two widely used schemes of PA-diagnostics are then directly compared. The time-resolution of the PA-diagnostics in analysis of simultaneous bi-exponential decay is evaluated, and the relationship between the resolving power and parameters of the experimental set-up (signal-to-noise ratio, sampling rate, shape of the PA-filter) is found. The advantage of front face irradiation scheme with piezopolymer film detector for time-resolved measurements is demonstrated.     

Characterization of VDAC1 as a plasma membrane NADH-oxidoreductase

We have recently demonstrated that voltage dependent anion selective channel~1 (porin, isoform 1) can function as a transplasma membrane NADH:ferricyanide-reductase. However, both the specific redox characteristics and the mechanism of electron transport in this enzyme presently remain unclear. Here we demonstrate that the redox capability of porin 1 is specific for ferricyanide as this same enzyme cannot reduce DCIP or cytochrome c in vitro. Furthermore, NADH-dependent ferricyanide reduction associated with VDAC1 is not sensitive to the anion channel inhibitors DIDS and dextran sulfate. However, this activity can be inhibited by thiol chelators, suggesting that at least one of the two cysteine groups present in VDAC1 are critical for electron transfer. We propose a model on how electron transport may occur in VDAC1.     

Simple formal kinetics for the reversible uptake of molecular hydrogen by [Ni-Fe] hydrogenase from Desulfovibrio gigas.

Enzymatic electrocatalysis, triggered and monitored by means of cyclic voltammetry, enabled us to achieve quantitative analysis of the kinetics of the hydrogenase catalyzed process, in the 7.8-10.0 pH range, in the presence of an electrochemically generated redox mediator. The quantitative analysis can be carried out by use of a quite simple SRC model. The simplicity of the SRC model is compatible with the existence of multiple redox microstates, which can be combined in a potential adjustable triangular mechanism consisting of three catalytic cycles, which are formally identical from the kinetic point of view. The steps involved in the kinetic control of the reversible process are H2 uptake or production at the Ni-Fe catalytic site and the intermolecular electron transfer between the mediator and the distal [4Fe-4S] cluster. The related rate constants have been determined. For the two accompanying intramolecular electron transfers which proceed at equilibrium, the equilibrium constants were found to be in very good agreement with previously published data.     

Effect of the synergistic anion on electron paramagnetic resonance spectra of iron-transferrin anion complexes is consistent with bidentate binding of the anion.

Continuous wave (cw) X-band EPR spectra at approximately 90 K were obtained for iron-transferrin-anion complexes with 18 anions. Each anion had a carboxylate group and at least one other polar moiety. As the second polar group was varied from hydroxyl to carbonyl to amine to carboxylate, the EPR spectra changed from a dominant signal at g' approximately 4.3 with a second smaller peak at g' approximately 9 to a broad signal with intensity between g' approximately 5 and 7. Computer simulation indicated that the changes in the EPR spectra were due to changes in the zero field splitting parameter ratio, E/D, from approximately 1/3 for carbonate anion to approximately 0.04 for malonate anion. Observation of iron-13C coupling in the electron spin echo envelope modulation (ESEEM) for iron transferrin [1-13C]pyruvate indicated that the carboxylate group was bound to the iron. It is proposed that all of the anions behave as bidentate ligands, with coordination to the iron through both the carboxylate and proximal groups, and the carboxyl group serves as a bridge between the iron and a positively charged group on the protein.     

Use of stopped-flow spectrophotometry to establish midpoint potentials for redox proteins

Stopped-flow spectrophotometry was examined as a tool to assign midpoint potentials to protein redox half-reactions. The method involves the rapid mixing of protein and electron transfer mediator solutions and the determination of the absorbance of at least one of the reacting species or products at equilibrium. The utility of the method was demonstrated with two different redox proteins (nitrogenase iron protein and cytochrome c) with very different midpoint potentials. The overall errors ranged from about +/-0.5 to 3 mV. Advantages of the method include short times required for the experiments, the precision and accuracy of the method in comparison to other methods, the conservative use of valuable protein in the experiments and the ease of obtaining midpoint potentials for redox protein half-reactions, and the potential range covered by a single electron transfer mediator when the method involves mediated electron transfer. It is concluded that the stopped-flow spectrophotometry should be considered the method of choice for determining protein midpoint potentials.