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The detailed process of the hydrolysis of ferricyanide into dendritic α-Fe2O3 (hematite) crystals with snowflake-like, feather-like, and leaf-like morphologies has been investigated. [Fe(CN)6]3- anions were found to polymerize into large, disordered soft matter aggregates at an early stage. The nucleation of hematite crystals took place near the surface of these aggregates via further hydrolysis. After the crystals grew to a certain size, branches started to appear. When the concentration of ferricyanide was low (i.e. 2 mM to 3.8 mM), growth was preferentially along the six equivalent ⟨112̅0⟩ directions, resulting in a flat snowflake-like shape, while high concentrations (i.e. 9 mM to 500 mM) of ferricyanide led to the growth of selective directions along the ⟨101̅1⟩ zone axes, forming a feather-like or leaf-like morphology. Highly selective adsorption and surface hydrolysis of [Fe(CN)6]3- anions on α-Fe2O3 crystals was found to be a crucial process in the formation of these novel morphologies. It was found that the polymerization of ferricyanide led to a reduction of pH value and that the formation of Fe2O3 increased the pH value. The pH value of the solution at the point when the branches start to grow can significantly affect the distribution of Lewis acidic sites on different surfaces and, therefore, change the growth direction. The newly established mechanism is complementary to the classical theories of crystal growth. [ABSTRACT FROM AUTHOR]

Here we report a novel solid-state ECL sensor for ultrasensitive sensing of glutathione (GSH) based on ferrocyanide-ferricyanide redox couple (Fe(CN) 63-/4- ) induced electrochemiluminescence (ECL) amplification of carbon dots (C-dots). The electropolymerization of C-dots and (11-pyrrolyl-1-yl-undecyl) triethylammonium tetrafluoroborate (A2) enabled immobilization of the hydrophilic C-dots on the surface of glassy carbon electrode (GCE) perfectly, while the excellent conductivity of polypyrrole was exploited to accelerate electron transfer between them. The Fe(CN) 63-/4- can expeditiously convert the C-dots and S2O82- to C-dot and SO4, respectively. High yields of the excited state C-dots (C-dots) were obtained, and a ~10-fold ECL amplification was realized. The C-dots* obtained through the recombination of electron-injected and hole-injected processes may be impeded due to the interference of GSH to K2S2O2. Therefore, the constructed sensor for GSH showed a detection limit down to 54.3 nM (S/N = 3) and a wide linear range from 0.1-1.0 μM with a correlation coefficient of 0.997. [ABSTRACT FROM AUTHOR]

Theincorporation and electrochemical activity of a conjugatedoligoelectrolyte (COE) in model phospholipid bilayers have been characterizedusing cyclic voltammetry and UV–vis absorption measurements.Several other modifiers were also incorporated into the phospholipidmembranes to alter properties such as charge and alkyl chain disorder.Using potassium ferricyanide to measure charge transport, it was observedthat bilayers that contained cholic acid, a negatively charged additivethat also promotes alkyl chain disorder, had higher COE uptake andcharge permeability than unmodified bilayers. In contrast, when thepositively charged choline was incorporated, charge permeability decreasedand COE uptake was similar to that of unmodified bilayers. The incorporationof cholesterol at low concentrations within the phospholipid membraneswas shown to enhance the COE’s effectiveness at increasingmembrane charge permeability without increasing the COE concentrationin the bilayer. Higher concentrations of cholesterol reduce membranefluidity and membrane charge permeability. Collectively, these resultsdemonstrate that changes in phospholipid membrane charge permeabilityupon COE incorporation depend not only on the concentration in themembrane but also on interactions with the phospholipid bilayer andother additives present in the membranes. This approach of manipulatingthe properties of phospholipid membranes to understand COE interactionsis applicable to understanding the behavior of a wide range of moleculesthat impart useful properties to phospholipid membranes. [ABSTRACT FROM AUTHOR]

Combined computationaland experimental techniques were employedto investigate at the microscopic level the structural and dynamicproperties of ferro- and ferricyanide ions in aqueous solution. Thecharacterization of the structural patterns and multiscale dynamicstaking place within the first solvation spheres in water and heavywater solvents was first achieved through extensive molecular dynamicssimulations, performed with refined force fields, specifically parametrizedfor the cyanide ions under investigation. The information gained aboutthe solute–solvent interactions is then validated through thesuccessful comparison of computed and measured waiting-time-dependent2D IR spectra. The vibrational patterns resulting from 2D IR measurementswere rationalized in terms of the interaction between the ion andthe neighboring water molecules described by simulation. It was foundthat, within the first solvation sphere, the stronger interactionsof the solvent with the ferro species are responsible for a delayin the relaxation dynamics, which becomes more and more evident onlonger time scales. [ABSTRACT FROM AUTHOR]

Recently, we introduced a new electrochemical imaging technique called fluorescence-enabled electrochemical microscopy (FFEM). The central idea of FEEM is that a closed bipolar electrode is utilized to electrically couple a redox reaction of interest to a complementary fluorogenic reaction converting an electrochemical signal into a fluorescent signaL This simple strategy enables one to use fluorescence microscopy to observe conventional electrochemical processes on very large electrochemical arrays. The initial demonstration of FEEM focused on the use of a specific fluorogenic indicator, resazurin, which is reduced to generate highly fluorescent resorufin. The use of resazurin has enabled the study of analyte oxidation reactions, such as the oxidation of dopamine and H2O2. In this report, we extend the capability of FEEM to the study of cathodic reactions using a new fluorogenic indicator, dihydroresorufin. Dihydroresorufin is a nonfluorescent molecule, which can be electrochemically oxidized to generate resorufin. The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)63- and Ru(NH3)63+. Here we demonstrate the correlation between the simultaneously recorded fluorescence intensity of resorufin and electrochemical oxidation current during potential sweep experiments. FEEM is used to quantitatively detect die reduction of ferricyanide down to a concentration of approximately 100 μM on a 25 pm ultramicroelectrode. We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significandy decreases diftusional broadening of the signal in FEEM as compared to resazurin. [ABSTRACT FROM AUTHOR]

In this work, we carried out steady-stateIR absorption, transientIR pump–probe, and waiting-time-dependent two-dimensional (2D)IR measurements on ferrocyanide and ferricyanide ions solvated inwater and deuterated water. These two anions are highly symmetricand have distributed cyano groups with IR-active stretching modesin the 5 μm wavelength region. The line width of their linearIR spectra and the initial value of the vibrational frequency–frequencycorrelation function extracted from their 2D IR spectra indicate watermolecules in the hydration shell of the ferro-species are more inhomogeneouslydistributed but more tightly bound to the cyano groups than thoseof the ferri-species. Different charges and their distributions inthe two anions cause different hydrogen bonding strengths with solvent.The frequency correlation relaxes somewhat slower in ferrocyanide,agreeing with stronger solute–solvent hydrogen-bonding interactionin this case. Mechanisms of the solvent isotope effect on the vibrationalrelaxation dynamics of the cyano stretching mode are discussed. Theseresults also suggest that in the hydration shell the ferro-speciesbreaks more water structure than the ferri-species, which is oppositeto the situation of the bulk water region (beyond the hydration shell)reported previously. This work demonstrated that combined IR methodscan be very useful for understanding the molecular details of thestructure and dynamics of the hydrated ions. [ABSTRACT FROM AUTHOR]

Data from Kα resonant inelastic X-ray scattering (RIXS) have been used to extract electronic structure information, i.e., the covalency of metal–ligand bonds, for four iron complexes using an experimentally based theoretical model. Kα RIXS involves resonant 1s→3d excitation and detection of the 2p→1s (Kα) emission. This two-photon process reaches similar final states as single-photon L-edge (2p→3d) X-ray absorption spectroscopy (XAS), but involves only hard X-rays and can therefore be used to get high-resolution L-edge-like spectra for metal proteins, solution catalysts and their intermediates. To analyze the information content of Kα RIXS spectra, data have been collected for four characteristic σ-donor and π-back-donation complexes: ferrous tacn [FeII(tacn)2]Br2, ferrocyanide [FeII(CN)6]K4, ferric tacn [FeIII(tacn)2]Br3 and ferricyanide [FeIII(CN)6]K3. From these spectra metal–ligand covalencies can be extracted using a charge-transfer multiplet model, without previous information from the L-edge XAS experiment. A direct comparison of L-edge XAS and Kα RIXS spectra show that the latter reaches additional final states, e.g., when exciting into the eg (σ*) orbitals, and the splitting between final states of different symmetry provides an extra dimension that makes Kα RIXS a more sensitive probe of σ-bonding. Another key difference between L-edge XAS and Kα RIXS is the π-back-bonding features in ferro- and ferricyanide that are significantly more intense in L-edge XAS compared to Kα RIXS. This shows that two methods are complementary in assigning electronic structure. The Kα RIXS approach can thus be used as a stand-alone method, in combination with L-edge XAS for strongly covalent systems that are difficult to probe by UV/vis spectroscopy, or as an extension to conventional absorption spectroscopy for a wide range of transition metal enzymes and catalysts. [ABSTRACT FROM AUTHOR]

The development of spectroscopic and electrochemical devices that can accommodate very small samples is of considerable importance to many areas of science and technology. We report here on the design and characteristics of a simple apparatus for the electrochemical and spectroelectrochemical analysis of submicroliter aqueous samples. The device is easily assembled from common laboratory materials and equipment, utilizing a bifurcated fiber-optic probe, standard disk electrodes of millimeter dimensions, and a polymer electrolyte film salt bridge to enable the analysis of nanoliter-scale sample volumes in a thin-layer configuration. Excellent performance has been demonstrated via measurements on aqueous ferricyanide solutions using sample volumes as low as 20 nL. [ABSTRACT FROM AUTHOR]

Water oxidation by plants and cyanobacteria is performed via a light-driven cycle of five intermediates called S states (S0-S4) at the water oxidizing center (WOC) in photosystem II (PSII). The information about misses, i.e., the probabilities that the S-state transitions failed to advance, is crucial for detailed analysis of various spectroscopic data in investigations of the water oxidation mechanism. In this study, we have determined the miss probabilities of the individual S-state transitions using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The extent of S-state transitions in the WOC upon each saturating flash was monitored by detecting the flow of electrons from the WOC to ferricyanide, an exogenous electron acceptor, using the CN stretching bands of ferricyanide and ferrocyanide. Simulation of the oscillation pattern of the flash-number dependence of the signal amplitude provided the miss probabilities for the S0 → S1, S1 → S2, S2 → S3, and S3 → S0 transitions (α0-α3, respectively) without any assumption about fitting parameters. The results for PSII preparations from Thermosynechococcus elongatusand spinach showed a general tendency of misses in the order, α0 ⩽ α1 < α2 < α3, indicating that a more oxidized WOC has a higher miss probability. A very similar result observed for the YD-less mutant (D2-Y160F) of T. elongatus confirmed that YD does not affect the estimated misses. It was further shown that NO3- treatment specifically increased α3, consistent with inactivation of the S3 state reported previously. These results demonstrate the usefulness of this FTIR method for estimating individual miss probabilities in the S-state cycle in elucidation of the molecular mechanism of photosynthetic water oxidation. [ABSTRACT FROM AUTHOR]

The redox reactions of organic radicals, with Fe(CN)63–and methylviologen, generated from the sonochemical decomposition of aliphaticalcohols in aqueous solutions, have been studied. The number of radicalsproduced was found to relate to the amount of adsorbed alcohol molecules(Gibbs surface excess) at the gas–aqueous solution interfacefor any bulk solution concentration of the alcohol. The majority ofthe radicals produced stem from the thermal degradation of the alcoholmolecules that have entered imploding cavitation bubbles. The maximumrate of reduction at 355 kHz, of Fe(CN)63–, was 2.6 ± 0.3 μMmin–1, whereas for methyl viologen, it was 1.2 ±0.3 μM min–1under the conditions used. Thedifference in the rates is attributed to the reaction of various pyrolyticallyproduced organic radicals with the methyl viologen radical cation.The possible reactions occurring in the sonolysis of alcohol/watersystems are discussed in detail. [ABSTRACT FROM AUTHOR]

This study effectively demonstrates a strategy to enable the ferricyanide-based second-generation biosensors for selective in vivo measurements of neurochemicals, with glucose as an example. The strategy is based on regulation of redox potential of ferricyanide mediator by carefully controlling the different adsorption ability of ferricyanide (Fe(CN)63-) and ferrocyanide (Fe(CN)64-) onto electrode surface. To realize the negative shift of the redox potential of Fe(CN)63-/4-, imidazolium-based polymer (Pim) is synthesized and used as a matrix for surface adsorption of Fe(CN)63-/4- due to its stronger interaction with Fe(CN)63- than with Fe(CN)64-. The different adsorption ability of Fe(CN)63- and Fe(CN)64- onto electrodes modified with a composite of Pim and multiwalled carbon nanotubes (MWNT5) eventually enables the stable surface adsorption of both species to generate integrated biosensors and, more importantly, leads to a negative shift of the redox potential of the surface-confined redox mediator. Using glucose oxidase (GOD) as the model biorecognition units, we demonstrate the validity of the ferricyanide-based second-generation biosensors for selective in vivo neurochemical measurements. We find that the biosensors developed with the strategy demonstrated in this study can be used well as the selective detector for continuous online detection of striatum glucose of guinea pigs, by integration with in vivo microdialysis. This study essentially paves a new avenue to developing electrochemical biosensors effectively for in vivo neurochemical measurements, which is envisaged to be of great importance in understanding the molecular basis of physiological and pathological events. [ABSTRACT FROM AUTHOR]

Electrochemical reactions are normally initiated in solution by metal electrodes such as Pt, which are expensive and limited in supply. In this Communication, we demonstrate that an atmospheric-pressure microplasma can act as a gaseous, metal-free electrode to mediate electron-transfer reactions in aqueous solutions. Ferricyanide is reduced to ferrocyanide by plasma electrons, and the reduction rate is found to depend on discharge current. The ability to initiate and control electrochemical reactions at the plasma-liquid interface opens a new direction for electrochemistry based on interactions between gas-phase electrons and ionic solutions. [ABSTRACT FROM AUTHOR]

Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (kcat = 2.0 s-1; kcat/Km = 2.5 x 103 M-1s-1). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s-1 and 5 x l05 M-1 s-1, respectively. The apoenzyme was prepared and reconstituted with Fe2+, Zn2+, or Mn2+. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [FeIIFeII ]-ADE was oxidized to [FeIII/FeIII]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [FeIII/FeIII]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. t4o evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Mössbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 A resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate-limiting steps. [ABSTRACT FROM AUTHOR]

Cation free radicals of bacteriochlorophyll (BChl+) are formed in the light harvesting complex 1 (LH1) of photosynthetic bacteria upon oxidation by potassium ferricyanide. Unusually narrow EPR line widths are observed for BChl+in the frozen state. These narrow line widths are consistent with a molecular-wire behavior where rapid electron/hole transfer occurs between the BChl constituents of the pigment array responsible for light harvesting in bacterial photosynthesis. However, in addition to electron/hole transfer, two distinct types of spin−spin exchange could contribute the EPR line width narrowing, thus obfuscating the determination of LH1 as a molecular wire. First, because excess ferricyanide ion is always present during the EPR measurements, electron spin−spin interactions between the paramagnetic ferricyanide and BChl+could be a major source of the EPR line width changes previously attributed solely to electron/hole hopping within the array of BChl molecules in a LH1 unit. Fixing the potential of the ferricyanide/ferrocyanide redox couple gives a constant concentration of paramagnetic iron as the amount of BChl oxidized in LH1 changes. As long as the fraction of oxidized BChl in LH1 remains the same, the EPR line width is found independent of the concentration of the ferricyanide oxidant. Additionally, the trend in EPR line width as a function of temperatures depends only on the fraction of oxidized BChl and not on the concentration of ferricyanide ion. Second, spin−spin exchange interactions between BChl+s within LH1 rings could also change the EPR line width. Using LH1 preparations containing at most a few BChl cations per LH1 complex also eliminates the occurrence of significant electron spin−spin exchange as a cause of the observed line width narrowing in minimally oxidized LH1. This investigation of the two types of electron spin−spin exchange interactions demonstrates (1) that electron/hole hopping can take place in oxidized LH1 without involvement of paramagnetic ferricyanide or spin−spin exchange between BChl+s and (2) that LH1 maintains a molecular-wire nature at cryogenic temperatures. [ABSTRACT FROM AUTHOR]

Nostoc sp. (Na) H-NOX is a heme protein found in symbiotic cyanobacteria, which has 35% sequence identity and high structural homology to the fi subunit of soluble guanylyl cyclase (sGC), suggesting a NO sensing function. However, UV-vis. EPR. NIR MCD. and ligand binding experiments with ferrous and ferric Ns H-NOX indicate significant functional differences between iVs H-NOX and sGC. (I) After NO binding to sGC. the proximal histidine dissociates from the heme iron, causing a conformational change that triggers activation of sGC. In contrast, formation of pentacoordinate (5c) NO heme occurs to only a limited extent inNs H-NOX, even at > I mM NO. (2) Unlike sGC, two different hexacoordinate (6c) NO complexes are formed in Na H-NOX with initial and final absorbance peaks at 418 and 414 nm. and the conversion rate is linearly dependent on [NO], indicating that a second NO binds transiently to catalyze formation of the 414 nm species. (3) sGC is insensitive to oxygen, and ferric sGC prepared by ferricyanide oxidation has a 5c high-spin heme complex. In contrast, Na H-NOX autoxidizes in 24 h if exposed to air and forms a 6c ferric heme complex, indicating a major conformational change after oxidation and coordination by a second histidine side chain. Such a large conformational transition suggests that Ns H-NOX could function as either a redox or a NO sensor in the cyanobacterium. [ABSTRACT FROM AUTHOR]

The diffusion-controlled electron transfer rate constants (kd) of several quenching reactions of ruthenium complexes [Ru(L)3]2+*(L = bpy, phen, and 4,7-(CH3)2phen) with [Fe(CN)6]3−were experimentally determined at different concentrations of NaNO3. From these rate constants, the effective values of viscosity coefficients for NaNO3solutions were calculated using EMSA (exponential mean spherical approximation) and EF (Eigen-Fuoss) approaches in order to take into account the mean force potential between reactants. The reliability of the effective parameters were checked through calculations of the rate constants of the reaction [IrCl6]2−+ [Ru(bpy)3]2+* in these NaNO3solutions. The rate constants of this reaction were also obtained by fluorescence quenching measurements. The agreement between the two sets of data (experimental and predicted) is excellent. The trends of association (kd) and dissociation (k−d) rate constants for 2+/3−, 2+/2−, and 2+/2+ reactions in NaNO3solutions are discussed. The use of effective diffusion coefficients for estimating kdand k−dallowed us to obtain the intrinsic electron transfer rate constant (ket) for the activation-diffusion-controlled process between [Ru(bpy)3]2+* and [Co(NH3)5Cl]2+complexes from the observed (quenching) rate constant. The trend of electron-transfer rate constant in NaNO3for this reaction was rationalized by using the Marcus electron-transfer treatment. [ABSTRACT FROM AUTHOR]

Cyclic biamperometry has been investigated as a method for the quantitation of one form of a reversibly electroactive redox couple in the presence of the other form, using the ferri-ferrocyanide couple in aqueous KCI. A triangular voltage waveform applied across two equal-area, planar gold electrodes yields peak currents that depend on the square root of the applied voltage scan rate, when one form of the redox couple is present in excess. Independent measurement of electrode-to-solution potential during biamperometric scans allowed estimation of the fractional impedance at each electrode-solution interface, and these values allow calculation of the effective potential scan rates at each electrode. Results show that when one form is present in a 5-fold excess or greater, the potential scan rate for the limiting reaction is nearly identical to the applied voltage scan rate. Similar values were obtained from impedance calculations, but discrepancies between the predicted and experimental values are evident when the two forms are present at near equivalent concentrations. When one form of the redox couple is present in excess, cyclic biamperometric peak currents depend linearly on the concentration of the limiting form, and these currents can be amplified by using cells with one electrode much larger than the other. Because this method does not require a reference electrode, it can, in principle, be readily incorporated into new electrochemical array or lab-on-a-chip devices. [ABSTRACT FROM AUTHOR]

NADH:ubiquinone oxidoreductase (complex I) is a complicated respiratory chain enzyme that conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the mitochondrial inner membrane. Alternatively, NADH oxidation, by the flavin mononucleotide in complex I, can be coupled to the reduction of hydrophilic electron acceptors, in non-energy-transducing reactions. The reduction of molecular oxygen and hydrophilic quinones leads to the production of reactive oxygen species, the reduction of nicotinamide nucleotides leads to transhydrogenation, and "artificial" electron acceptors are widely used to study the mechanism of NADH oxidation. Here, we use a combined modeling strategy to accurately describe data from three flavin-linked electron acceptors (molecular oxygen, APAD+, and ferricyanide), in the presence and absence of a competitive inhibitor, ADP-ribose. Our combined ping-pong (or ping-pong-pong) mechanism comprises the Michaelis-Menten equation for the reactions of NADH and APAD+, simple dissociation constants for nonproductive nucleotide-enzyme complexes (defined for specific flavin oxidation states), and second-order rate constants for the reactions of ferricyanide and oxygen. The NADH-dependent parameters are independent of the identity of the electron acceptor. In contrast, a further flavin-linked acceptor, hexaammineruthenium(III), does not obey ping-pong-pong kinetics, and alternative sites for its reaction are discussed. Our analysis provides kinetic and thermodynamic information about the reactions of the flavin active site in complex I that is relevant to understanding the physiologically relevant mechanisms of NADH oxidation and superoxide formation. [ABSTRACT FROM AUTHOR]

In this work, mediated amperometry was used to evaluate whether differences in intracellular nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) level could be observed between a genetically modified Saccharomyces cerevisiae strain, engineered for NADPH dependent 5-hydroxymethyl-2-furaldehyde (HMF) reduction, and its control strain. Cells overexpressing the alcohol dehydrogenase 6 gene (ADH6 strain) and cells carrying the corresponding control plasmid (control strain) were each immobilized on Au-microelectrodes. The real-time dynamics of NAD(P)H availability in the two strains, preincubated with HMF, was probed using the menadione-ferricyanide double mediator system. A lower intracellular NADPH level as the consequence of more effective HMF reduction was observed for the ADH6 strain both with and without added glucose, which increases the overall cellular NADPH level. The mediated amperometric signal during real-time monitoring of the concentration dependent HMF reduction in living cells could be translated into the cellular enzyme kinetic parameters: KappM,cell, VMAX, kcat,cell, and kcat,cell/KappM,cell. The results indicated that the overexpression of the ADH6 gene gave a 68% decrease in KappM,cell and 42% increase in VMAX, resulting in a 4-fold increase in kcat,cell/KappM,cell. These results demonstrate that the mediated amperometric method is useful for monitoring the short-term dynamics of NAD(P)H variations and determining cellular enzynne kinetic parameters in S. cerevisiae cells. [ABSTRACT FROM AUTHOR]

Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metabolism and is characterized by elevated levels of plasma homocysteine. In this study, we have examined the molecular basis of MSR dysfunction associated with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione reduction is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochemical penalty associated with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy. [ABSTRACT FROM AUTHOR]

Photoanode sensing platforms with remarkable photoelectrochemical (PEC) response and satisfying visible-light absorption have become the most promising detection systems. Nevertheless, their inevitable electrophilic character limits their expansion in the bioassay because of reductive substances in serum or other body fluids that can severely interfere with the photocurrent to be read. To solve it, a PEC platform-assembled dual-active electrode is designed to realize the separation of biological monitoring from the photoanode. The ferricyanide ([Fe(CN) 6 ] 3- )-mediated redox cycle is first proposed to meet the gain and loss electron requirements of the PEC system. It can avoid the self-reaction in the electrolyte caused by the addition of a traditional electron donor and acceptor, for instance, ascorbic acid and hydrogen peroxide. As a consequence, the traditional counter electrode (Pt wire) is replaced by Fe 2 O 3 /AgInS 2 heterojunction, which can amplify the PEC response of the cathode to meet the requirement of trace analysis. An aptasensor fabricated by the above strategies exhibits convincing data for 17β-estradiol (E2) detection from which a wide detection range is obtained in 10 fg/mL to 1 μg/mL with a detection limit of 2.74 fg/mL ( S / N = 3). These advanced elements show a rosy prospect for environmental monitoring and point-of-care biomarker diagnosis.

An electrochemical molecular sensor based on a new principle is reported. Nereistoxin (NRT, 4-N,N-dimethylamino-1,2-dithiolane), a naturally occurring neurotoxin (nicotinic acetylcholine receptor agonist), was adsorbed on an Au electrode via Au-S covalent bonding and accelerated the electron transfer between the electrode and the marker, ferricyanide anion. The contrast between the electrochemical responses obtained with the bare and NRT-modified Au electrodes was more pronounced at a low ionic strength of the supporting electrolyte, KCl. In the presence of 1 mM KCl, almost a 0/1 contrast between the signals was obtained through electrostatic interaction between the protonated tertiary amino group of NRT and the anionic ferricyanide ion. No current was observed with an electrode modified with mercaptopropionic acid. An unusually low ionic strength thickened the electric double layer to the degree where current was not observed with the bare electrode. The effect of the electrostatic concentration of the marker ion becomes obvious under such conditions. Commercially available NRT-related pesticides such as Cartap and Bensultap were also detected using the same format after pretreatments by hydrolysis/reduction. The present sensing method was successfully applied to human serum with satisfactory sensitivity.

Given their putative role in chemoprevention, validated methods are needed for quantification of total glucosinolates. Based on the colorimetric reaction of ferricyanide with 1-thioglucose, released by alkaline hydrolysis of glucosinolates, we developed a simple and sensitive method for spectrophotometric quantification of total glucosinolates in cruciferous vegetables. Lyophilized and ground vegetables are extracted with 80% boiling methanol. Extracted glucosinolates are isolated using a strong anion exchange column and then hydrolyzed with 2 N NaOH to release 1-thioglucose. Ferricyanide is added, and the decrease in absorbance is measured at 420 nm, with final values adjusted for background. Recovery of internal standard (sinigrin) was 107%. Intra- and interassay coefficients of variation were 5.4% and 15.8%, respectively. Dose response was linear with sinigrin and amount of plant material extracted (R(2) ≥ 0.99). Using sinigrin, the lower limit of quantification was 0.6 mg. This straightforward method may be an alternative to time-consuming and costly chromatographic methods.

The unfolding of the CuA domain of cytochrome oxidase from the thermophilic bacterium Thermus thermophilus, induced by guanidine hydrochloride (GuHCl)1 at different temperatures, has been monitored by CD as well by electronic absorption (with the oxidized protein) and by fluorescence (with the reduced protein). The same unfolding curves were obtained with the different methods, providing evidence for a two-state model for the unfolding equilibrium. This was also supported by the shape of the unfolding equilibrium curves and by the observed refolding of the unfolded, oxidized protein on dilution of the denaturant. The oxidized protein cannot be unfolded by GuHCl at room temperature, and it was found to be thermally very stable as well, since, even in the presence of 7 M GuHCl, it is not fully unfolded until above 80 degrees C. For the reduced protein at room temperature, the unfolding equilibrium curve yielded a folding free energy of -65 kJ/mol. The corresponding value for the oxidized protein (-85 kJ/mol) could be estimated indirectly from a thermodynamic cycle connecting the folded and unfolded forms in both oxidation states and the known reduction potentials of the metal site in the folded and unfolded states; the potential is increased on unfolding, consistent with the higher folding stability of the oxidized form. The difference in folding stability between the oxidized and reduced proteins (20 kJ/mol) is exceptionally high, and this is ascribed to the unique structure of the dinuclear CuA site. The unfolded, reduced protein was found to refold partially on oxidation with ferricyanide.

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.

The kinetics of cyanide binding to cytochrome c oxidase were systematically studied as a function of [HCN], [oxidase], pH, ionic strength, temperature, type and concentration of solubilizing detergent, and monomer-dimer content of oxidase. On the basis of these results a minimum reaction mechanism is proposed in which the spectrally visible rapid and slow cyanide binding reactions are two consecutive first-order reactions, not parallel reactions with different conformers of cytochrome c oxidase. The fast reaction (k'obs) follows saturation type kinetics to form an HCN complex that subsequently undergoes a slow reaction (k'obs). The fast k'obs reaction is independent of ionic strength but is strongly dependent upon pH. Two pK values were evaluated from the bell-shaped rate versus pH profile; one is due to an ionizable group on the protein (pKa = 7.45), while the other is that of HCN (pKHCN = 9.15). Therefore, oxidase is reactive toward HCN only when the group on the protein is unprotonated. The slow k'obs reaction is not a reaction of oxidase with either CN- or HCN; in fact, the product formed by the fast k'obs reaction, the oxidase-HCN complex, still undergoes the slow k" process even if all of the excess KCN is removed. The apparent rate constant of the slower phase (k"obs) is independent of all the variations done in this study, and it probably corresponds to either a slow conformational change in the protein or a change in ligand coordination at one of the metal centers after HCN binds to the bimetallic center of oxidase. Based upon the bell-shaped pH dependence of the fast phase and the pH independence of the slow phase, the mechanism also predicts that a single conformer of cytochrome c oxidase can exhibit either monophasic or biphasic cyanide binding kinetics depending upon the pH. At either very low or very high pH, the two rates become comparable in magnitude, which makes the reaction appear to be monophasic even though both reactions still occur. The amount of monomeric or dimeric oxidase only slightly affects the magnitude of k'obs and k"obs values, and both processes are clearly present in both types of oxidase.

The regulation by the proton gradient of the electron flow from water to ferricyanide was investigated in thylakoids extracted from lettuce leaves. When the transmembrane proton current was varied by an uncoupler or by the ATP synthase activity, a unique relationship was found between the rate of ferricyanide reduction and the proton gradient, restricted here to its delta pH component. This behavior was conserved in CF1-depleted thylakoids where the passive proton flow was varied by the concentration of an Fo inhibitor or by the concentration of an uncoupler after 100% inhibition of Fo. This shows that under our experimental conditions no direct proton transfer exists in steady state between the site of regulation of the redox chain and the ATPase. Studies at two different pH's indicate that the internal pH, and not the transmembrane pH difference, controls the electron transfer between PS2 and PS1. Modeling the data suggests that a single deprotonation step is kinetically limiting.

A recently introduced biosensor comprising a rotating bioreactor and a stationary platinum ring amperometric detector (Anal. Chem. 1993, 65, 636-639) has been utilized for the simultaneous determination of fructose and ascorbate. The approach has been successfully applied to the simultaneous determination of these analytes in fresh food samples. Hexacyanoferrate(II) is the monitored species at +0.380 V vs a Ag/AgCl, 3 M NaCl reference. The determination takes advantage of a fast chemical oxidation of ascorbate by hexacyanoferrate(III) ions and the subsequent slower production of hexacyanoferrate(II) in the D-fructose 5-dehydrogenase-catalyzed reaction between D-fructose and hexacyanoferrate(III) as acceptor. D-Fructose 5-dehydrogenase (EC 1.1.99.11) was incorporated into the rotating disk reactor in immobilized form and on controlled-pore glass via the glutaraldehyde attachment and cross-linking with bovine serum albumin. Sample/reagent processing was accomplished by programmed continuous-flow/stopped-flow/continuous-flow operation.

Absorbance and fluorescence methods, circular dichroism, UV melting experiments, viscosity, and competition dialysis were used to study the interaction of delta and lambda tris(phenanthroline)ruthenium(II) with DNA. The results of these studies indicated that both isomers bind to DNA by a single mode. The two isomers differ, however, in their effect on the hydrodynamic properties of DNA as measured by viscosity and, therefore, probably differ in their individual binding modes. The optical properties of the fully bound compounds differ from those of the free, but the perturbations of their visible absorbance and fluorescence emission spectra are modest when compared to changes observed for other DNA binding compounds. Binding of both isomers to DNA was found to be weak (in comparison to proven intercalators), with binding constants on the order of 10(4) M-1 determined for their binding to calf thymus DNA. A small, positive enthalpy was found for the binding of each isomer to DNA, suggesting that binding is entropically driven. Both isomers increased the melting temperature of DNA, with little quantitative difference between the two. A modest base specificity was found for each isomer, with the delta isomer preferentially binding to GC base pairs, and the lambda isomer preferentially binding to AT base pairs. Competition dialysis was used to examine the preference of delta and lambda Ru for right-handed B DNA and left-handed Z DNA. Neither isomer exhibits significant selectivity for these radically different DNA secondary structures.

Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(III)-EDTA, or peroxidase/H2O2, while phenelzine cleaved in the presence of Cu(II). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(III)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical (.OH) on the DNA damage suggest the involvement of .OH. On the other hand, Cu(II)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, but not by .OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(I) plus H2O2 but not to that induced by Cu(II) plus H2O2, it is speculated that the copper-oxygen complex derived from the reaction of H2O2 with Cu(I) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II). A comparison between scavenger effects on the DNA damage and those on radical production detected with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(II)-, hemin-, or Fe(III)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, .OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.

The electron-transfer mechanism of the Fe4S4 high-potential iron-sulfur proteins (HiPIP's) was explored via a stopped-flow spectrophotometric kinetic study of the reduction of Chromatium vinosum and Rhodopseudomonas gelatinosa HiPIP's by both native and trinitrophenyllysine-13 horse cytochrome c. The influence of electrostatic effects was also effectively partitioned from the redox process per se. The corrected rates were 12.3 X 10(4) and 3.8 X 10(4) M-1 s-1 for native with C. vinosum and R. gelatinosa HiPIP, respectively, and 17.5 X 10(4) and 5.46 X 10(4) M-1 s-1 for TNP-cytochrome c with the two HiPIP's, respectively. The faster rates of TNP-cytochrome c with the HiPIP's are unexpected in terms of possible steric interaction since lysine-13 is at the top of the heme crevice. In understanding the somewhat faster rates of the TNP-cytochrome c over native cytochrome c it is possible that (1) TNP-cytochrome c reacts more quickly since modification of the lysine-13 residue destabilizes somewhat the heme crevice or (2) in light of the hydrophobic nature of the trinitrophenyl group and the X-ray crystallographic structure of HiPIP, the TNP group facilitates electron transfer by interacting with a hydrophobic region on the HiPIP molecular surface. The region about the S4 sulfur atom is the most exposed and accessible hydrophobic region on the HiPIP surface, in addition to being the point of closest approach of the S4 to the external environment.

Direct chemical titrations of reduced, purified cytochrome aa3 were monitored at 604 nm. Anaerobic oxidation by potassium ferricyanide or 1,1'-bis(hydroxymethyl)-ferricinium ion was compared with the natural substrate, molecular oxygen. The four electrons from reduced cytochrome aa3 were donated to one oxygen molecule, resulting in a linear titration with only fully oxidized or fully reduced enzyme molecules present. Unlike the linear or sigmoidal titrations which resulted from various reductive titrations reported previously, pronounced hyperbolic curves were obtained with the chemical oxidants. The midpoint potential values exhibited by the four metal centers of cytochrome aa3 were determined by computer simulation of the titration curves. A high potential pair of components (heme a = 340 mV, Cu = 340 mV) and a low potential pair (heme a = 220 mV, Cu = 240 mV) were observed, consistent with literature values. Unique to these equilibrium studies was a split in the heme a extinction coefficients at 604 nm. The low potential heme a component contributed 80-85% to the total absorbance change. A polarographic assay was used to verify that the integrity of cytochrome aa3 remained intact during equilibrium titrations spanning several hours. These experimental results indicate that simple chemical reversibility does not exist for cytochrome aa3 under anaerobic conditions.

The electron transfer reaction between the "blue" single copper protein azurin (from Pseudomonas aeruginosa) and the hexacyanoiron (II/III) couple has been studied. Equilibrium constants for the reduction of azurin were measured spectrophotometrically in the temperature range 5-33 degrees C (K = 1.1 X 10(-2) at 25 degrees C, deltaH degrees = 10.9 kcal/mol, 0.1 M potassium phosphate, pH 7.0, I = 0.22). The enthalpy change was also determined by microcalorimetry and from the analysis of chemical relaxation amplitudes. Following a temperature-jump perturbation of this equilibrium, only a single relaxation was observed. The reciprocal of the relaxation time increased linearly as oxidized azurin was reacted with increasing amounts of ferrocyanide, yet reached saturation when reduced azurin was titrated with ferricyanide. This behavior as well as the analysis of the relaxation amplitudes led to the following scheme for this system: see article. At 25 degrees C the rate constants for the electron transfer were k+3=6.4s-1 and k-3=45s-1, the association constants K1=54 M-1 and K2-1=610 M-1. The activation and overall thermodynamic parameters as well as the individual thermodynamic values for the different steps were combined to construct a self-consistent energy profile for the reaction.

Anaerobic oxidative titrations of purified cytochrome aa3 were monitored at three wavelengths (444, 604, and 820 nm), in both the absence and the presence of carbon monoxide. Computer simulation of each titration curve was utilized to ascertain the midpoint potentials of the four oxidation-reduction centers of the enzyme. For experiments performed under nitrogen, two components were found to titrate with low potential (heme aL = 220 mV, CuL = 240 mV) and two with high potential (heme ath, cuH = 340 mV), consistent with results obtained previously in reductive titrations. Unequal heme extinction coefficients were observed at 444 nm. Oxidation by either potassium ferricyanide or 1,1'-bis(hydroxymethyl)ferricinium ion showed that the low potential heme component contributed 75% of the absorbance change at 444 nm. At 820 nm, the entire absorbance change could be attributed to a single, low potential copper component. Midpoint potentials calculated for the carbon monoxide complexed enzyme agreed with previously reported values. The copper components retained the values observed under nitrogen, while the titratable heme group gave an apparent midpoint potential of 260 mV. These results enable us to assign absorbance changes at various wavelengths to specific redox components of cytochrome aa3.

Initial rate measurements were made of the oxidation of D-lactate and D-alpha-hydroxybutyrate by oxygen and potassium ferricyanide, catalyzed by D-lactate dehydrogenase from Megasphera elsdenii. The detailed kinetic work indicates a "ternary complex" type mechanism, with a complex of keto acid and reduced enzyme reacting with the electron acceptor at pH 8. However, as the pH is lowered, the double-reciprocal plots become nonlinear, with a downward curvature. This seems to be due to negative interactions within the protein rather than to a complexity of the kinetic mechanism. The variation of initial rate parameters at pH 8 with temperature yields nonlinear Arrhenius plots with a greater activation energy above the break point than below. This type of behavior has been previously reported only for fumarase (Massey, 1953). Studies with deuterated D-lactate show only a small isotope effect on phi 0 and phi 1 (KM/Vmax for lactate) but a large effect on phi 2 (Km/Vmax for ferricyanide).

The hemoprotein subunit (SiR-HP) of Escherichia coli NADPH-sulfite reductase contains one siroheme (high-spin Fe3+, D = 8 cm-1) and one oxidized Fe4S4 center per polypeptide. Christner et al. [Christner, J.A., Munck, E., Janick, P.A., & Siegel, L.M. (1981) J. Biol. Chem. 256, 2098-2101] have shown by Mossbauer spectroscopy that the two prosthetic groups of SiR-HP are magnetically exchange coupled in the oxidized enzyme, a result which indicates the presence of a chemical bridge between them. Photoreduction of SiR-HP in the presence of 5'-deazaflavin and ethylenediaminetetraacetic acid causes the enzyme to accept up to 2.0 electrons. The two reducible centers in SiR-HP are reduced independently with a midpoint potential difference of 65 mV, the siroheme being more positive. The first electron added to SiR-HP results in loss of the g = 6.63, 5.24, and 1.98 set of EPR signals due to the ferriheme and production of an EPR-silent state. The second added electron results in the parallel appearance of three distinct types of EPR signal: a novel species with g = 2.53, 2.29, and 2.07 (0.63 spin per heme); two "S = 3/2 type" species with g = 5.23, 2.80, and ca. 2.0 and g = 4.82, 3.39, and ca. 2.0 (together account for 0.16 spin per heme); and a very small amount of a "classical" reduced Fe4S4 center signal with g = 2.04, 1.93, and 1.91 (0.03 spin per heme). The temperature dependences of the "g = 2.29" and "g = 1.93" signals are similar to each other and are like those seen with other Fe4S4 center proteins. Addition of small amounts of guanidinium sulfate (0.1 M) to SiR-HP causes the spectrum of fully reduced enzyme to show primarily the S = 3/2 type species (g = 4.88, 3.31, and 2.08; 0.84 spin per heme), although the enzyme remains fully active. Optical spectral changes followed as a function of enzyme reduction show that marked changes occur in the Fe2+ siroheme optical spectrum when the Fe4S4 center becomes reduced or oxidized. These results indicate that the prosthetic groups of SiR-HP remain coupled when the enzyme is reduced. It is suggested that the novel EPR signals result from exchange interaction between S = 1 or 2 ferroheme and S = 1/2 reduced Fe4S4.

Ghosts from Escherichia coli have been oxidized with CuSO4-o-phenanthroline or ferricyanide-ferrocene. Upon oxidation they became resistant to boiling dodecyl sulfate. The resulting rod-shaped "oxidation containers" apparently held together by disulfide bridges, are practically pure protein. They are soluble in dodecyl sulfate when reduced and they contain a set of about 30 different polypeptide chains. The four major ghost membrane proteins are not represented among the "oxidation proteins." Comparison of data obtained from digestion of ghosts with trypsin or particle-bound trypsin showed that most of the "oxidation proteins" appear to be located at the outer surface of the ghost membrane which is derived from the outer cell envelope membrane. One of the major ghost membrane proteins, II, is partially digested by trypsin, and it is shown that its trypsin sensitive part is also exposed only at the outer surface of the ghost membrane. Native cells could be oxidized only with low yields of "oxidation containers." However, cell envelopes prepared without detergents or chelating agents, as well as cells depleted of phospholipid or treated with sucrose-Triton X-100, are completely accessible to oxidation. In each case, the same set of proteins as that present in "oxidation containers" from ghosts was found to be covalently linked. Treatment of cells with trypsin caused the loss of about five "oxidation proteins" and a complete loss of oxidizability of the ghosts derived from these cells. It therefore appears that arrangement and localization of the "oxidation proteins" are not greatly different in cells and in ghosts, i.e., that these proteins are also situated asymmetrically at the outer cell envelope membrane.