Changes In Reduction Potential Research Articles

Synthesis, Structure, and Reactivity of the [Fe4S4(SR)4]2-(R = 2-, 3-, and 4-Pyridinemethane) Clusters

The <TEX>$[Fe_4S_4]^{2+}$</TEX> clusters with 2-, 3-, and 4-pyridinemethanethiolate (S2-Pic, S3-Pic, and S4-Pic, respectively) terminal ligands have been synthesized from the ligand substitution reaction of the <TEX>$(^nBu_4N)_2[Fe_4S_4Cl_4]$</TEX> (I) cluster. The new <TEX>$(^nBu_4N)_2[Fe_4S_4(SR)_4]$</TEX> (R = 2-Pic; II, 3-Pic; III, 4-Pic; IV) clusters were characterized by FTIR and UV-Vis spectroscopy. Cluster II was crystallized in the monoclinic space group C2/c with a = 24.530 (5) <TEX>$\AA$</TEX>, b = 24.636(4) <TEX>$\AA$</TEX>, c = 21.762(4) <TEX>$\AA$</TEX>, <TEX>${\beta}=103.253(3)^{\circ}$</TEX>, and Z = 8. The X-ray structure of II showed two unique 2:2 site-differentiated <TEX>$[Fe_4S_4]^{2+}$</TEX> clusters due to the bidentate-mode coordination by 2-pyridinemethanethiolate ligands. Cluster III was crystallized in the same monoclinic space group C2/c with a = 26.0740(18) <TEX>$\AA$</TEX>, b = 23.3195(16) <TEX>$\AA$</TEX>, c = 22.3720(15) <TEX>$\AA$</TEX>, <TEX>${\beta}=100.467(2)^{\circ}$</TEX>, and Z = 8. The 3-pyridinemethanethiolate ligand of III was coordinated to the <TEX>$[Fe_4S_4]^{2+}$</TEX> core as a terminal mode. Cluster IV with 4-pyridinemethanethiolate ligands was found to have a similar structure to the cluster III. Fully reversible <TEX>$[Fe_4S_4]^{2+}/[Fe_4S_4]^+$</TEX> redox waves were observed from all three clusters by cyclic voltammetry measurement. The electrochemical potentials for the <TEX>$[Fe_4S_4]^{2+}/[Fe_4S_4]^+$</TEX> transition decreased in the order of II, III and IV, and the reduction potential changes by the ligands were explained based on the structural differences among the complexes. The complex III was reacted with sulfonium salt of <TEX>$[PhMeSCH_2-p-C_6H_4CN](BF_4)$</TEX> in MeCN to test possible radical-involving reaction as a functional model of the [<TEX>$Fe_4S_4$</TEX>]-SAM (S-adenosylmethionine) cofactor. However, the isolated reaction products of 3-pyridinemethanethiolate-p-cyanobenzylsulfide and thioanisole suggested that the reaction followed an ionic mechanism and the products formed from the terminal ligand attack to the sulfonium.

Study on Thiourea Dioxide’s Reducing Capacity and its Reactivity with Reactive Dyes

In this paper, potential analysis method was applied to study the effects of pH value and temperature on potential and reducing capacity of thiourea dioxide, and the reactivity between thiourea dioxide and reactive dyes as ground shade dyes under simulated printing steaming conditions in aqueous solution was also investigated. The results showed that thiourea dioxide’s reduction potential increased with the rise of pH value and temperature. when temperature was lower than 60 °C, thiourea dioxide had low reactivity with reactive dyes as ground shade dyes, and when temperature was up to 80 °C, thiourea dioxide began to break reactive dyes, which was in accordance with the reduction potential change rule of thiourea dioxide along with temperature.

Kinetic evolution studies of silver nanoparticles in a bio-based green synthesis process

Silver nanoparticles are being extensively studied due to their widespread applications and unique properties. In the present study, the growth kinetics of silver nanoparticles as synthesized on reduction of silver nitrate solution by aqueous extract of Azadirachta indica leaves was investigated. The formation of silver nanoparticles was preliminarily monitored by measuring the absorption maxima at different time intervals after adding the reducing agent to the silver salt solution (0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h). At different time points characterization studies were conducted using X-ray diffraction studies, FT-IR techniques, zeta potential studies and transmission electron microscopy. The total available silver in the reaction medium was determined at different durations using ICP-OES. The changes in reduction potential in the medium were also monitored using potentiometric analysis. The results confirm a definite change in the medium pertaining to formation of the stable nanoparticles after 2 h, and a significant increase in the agglomeration tendency after 4 h of interaction. The growth kinetic data of the nanoparticles till 3.5 h was found to fit the LSW model confirming diffusion limited growth.

The influence of aggregation on the redox chemistry of humic substances

Environmental context. The ability of humic substances (decaying plant and animal matter) to partake in redox reactions in the environment depends on the extent to which the various humic polymers aggregate in solution to form larger particles. This aggregation, in turn, is predicated on the solution conditions, especially ionic strength, the pH, and the types of cations present. Abstract. Aggregation and conformation play an important role in the aqueous redox chemistry of humic substances (HS). The reduction potentials of dissolved humic and fulvic acids vary with pH, ionic strength, and type of humate used, and depending on the solution conditions, they can abiotically reduce various species. Changes in HS reduction potential ranged from 60 to 140 mV on addition of divalent cations, whereas no significant changes were observed with equivalent additions of monovalent cations. Dynamic light scattering measurements showed that this behaviour paralleled the size changes obtained with humic aggregates under the same conditions. The effect was more pronounced at higher pH, where divalent cations caused a significant decrease in the average hydrodynamic radius, whereas monovalent cations did not. At pH 4, neither mono- nor divalent cations substantially affected aggregate sizes. Quinoid moieties, which are known to play an important role in the redox chemistry of HS, displayed fluorescence excitation–emission matrices with features related to changes in the reduction potential of HS. An increase in the reduction potential (Eh) induced by the addition of Ca2+, for instance, caused a red shift in the excitation–emission matrix maximum.

Reduction Potential Tuning of the Blue Copper Center in Pseudomonas aeruginosa Azurin by the Axial Methionine as Probed by Unnatural Amino Acids

The conserved axial ligand methionine 121 from Pseudomonas aeruginosa azurin (Az) has been replaced by isostructural unnatural amino acid analogues, oxomethionine (OxM), difluoromethionine (DFM), trifluoromethionine (TFM), selenomethionine (SeM), and norleucine (Nle) using expressed protein ligation. The replacements resulted in < 6 nm shifts in the S(Cys)-Cu charge transfer (CT) band in the electronic absorption spectra and < 8 gauss changes in the copper hyperfine coupling constants (AII) in the X-band electron paramagnetic resonance spectra, suggesting that isostructural replacement of Met resulted in minimal structural perturbation of the copper center. The slight blue shifts of the CT band follow the trend of stronger electronegativity of the ligands. This trend is supported by 19F NMR studies of the fluorinated methionine analogues. However, the order of AII differs, suggesting additional factors influencing AII. In contrast to the small changes in the UV-vis and EPR spectra, a large variation of > 227 mV in reduction potential was observed for the series of variants reported here. Additionally, a linear correlation was established between the reduction potentials and hydrophobicity of the variants. Extension of this analysis to other type 1 copper-containing proteins reveals a linear correlation between change in hydrophobicity and change in reduction potential, independent of the protein scaffold, experimental conditions, measurement techniques, and steric modifications. This analysis has also revealed for the first time high and low potential states for type 1 centers, and the difference may be attributable to destabilization of the protein fold by disruption of hydrophobic or hydrogen bonding interactions that stabilize the type 1 center.

Axial Methionine Has Much Less Influence on Reduction Potentials in a CuA Center than in a Blue Copper Center

The role of the highly conserved axial methionine of the purple CuA center in an engineered CuA azurin on modulating the reduction potentials of the copper center was investigated by a systematic replacement of the methionine with glutamate, aspartate, and leucine. In contrast to the same substitutions in the structurally related blue copper azurin, much smaller changes in reduction potential were observed in the CuA azurin upon replacing the methionine ligand with negatively charged Glu (-8 mV) and Asp (-5 mV) and more hydrophobic Leu (+16 mV). These findings are important in understanding the different roles of the two cupredoxins. The diamond core Cu2S2(Cys) structure of the CuA is much more resistant to variations of axial ligand interactions than the distorted tetrahedral structure of the blue copper protein. This difference may translate into a much wider range of reduction potentials (>1000 mV) for blue copper proteins that transfer electrons to a variety of partners in many different biological systems and a much narrower range of reduction potentials (<40 mV) for CuA proteins where a small difference in reduction potentials between the CuA and its redox partners is required.

Oxidation–Reduction Potential Changes in Aeration Tanks and Microprofiles of Activated Sludge Floc in Medium‐ and Low‐Strength Wastewaters

Real-time control of aeration tank operation is key to high-efficiency pollutant removal and energy savings. One of the aims of this study was to examine the potential for using redox potential (oxidation-reduction potential [ORP]) to indicate wastewater quality online in aeration tanks treating medium (chemical oxygen demand [COD] of 70 to 150 mg/L) and low (COD of 15 to 30 mg/L) pollutant-concentration wastewaters. The field-scale data provide a good relationship between ORP values and nutrient removal along the length of the aeration tanks. The ORP values increased dramatically as organic matter was removed along the aeration tanks, indicating the improvement of the bulk liquor redox status. Dissolved oxygen higher than 1.0 mg/L was necessary for good biodegradation and improvement of the liquid redox status. Nitrification occurred at higher ORP values (380 to 420 mV) than was the case for organic substrate oxidation (250 to 300 mV). The microprofiles obtained from microelectrode measurements substantiate the heterogeneity of the microbial processes inside activated sludge flocs. Because of microbial oxygen utilization, the aerobic region in the activated sludge floc was limited to the top layer (0.1 to 0.2 mm) of the activated sludge aggregate present in medium-strength wastewater, with an anoxic zone dominating inside the flocs. When dissolved oxygen in the bulk water was higher than 4.0 mg/L, the anoxic zone inside the floc disappeared. At low wastewater pollutant concentrations, the ORP and dissolved oxygen inside the activated sludge aggregates were higher than those from medium-strength wastewater. The prospect of using ORP as an online control approach for aeration tank operation and the potential reasons for activated sludge floc size varying with pollutant strengths are also discussed.

Prediction of Reduction Potential Changes in Rubredoxin: A Molecular Mechanics Approach

Predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment. Previously, we proposed that an alanine vs. a valine at residue 44 leads to a 50-mV difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes. Here, the aim is to determine the effects of mutations to glycine, isoleucine, and leucine at residue 44 on the structure and reduction potential of rubredoxin, and if the effects are proportional to side-chain size. Crystal structure analysis, molecular mechanics simulations, and experimental reduction potentials of wild-type and mutant Clostridium pasteurianum rubredoxin, along with sequence analysis of homologous rubredoxins, indicate that the backbone position relative to the redox site as well as solvent penetration near the redox site are both structural determinants of the reduction potential, although not proportionally to side-chain size. Thus, protein interactions are too complex to be predicted by simple relationships, indicating the utility of molecular mechanics methods in understanding them.

Synthesis, structure and properties of [Pt(2,2′-bipyridyl-5,5′-dicarboxylic acid)(3,4-toluenedithiolate)]: tuning molecular properties for application in dye-sensitised solar cells

The platinum diimine dithiolate complex, [Pt(2,2′-bipyridyl-5,5′-dicarboxylicacid)(3,4-toluenedithiolate)] ([Pt(5,5′-dcbpy)(tdt)]) and its tetrabutylammonium salt [TBA]2[Pt(5,5′-dcbpy)(tdt)] have been prepared, spectroscopically and electrochemically characterised and attached on to TiO2 substrate to be used as solar cell sensitisers. A single-crystal X-ray structure was obtained for [TBA]2[Pt(5,5′-dcbpy)(tdt)]·EtOH·EtOAc. The effect of the position of the two carboxylic acid substituents on the electrochemistry of the 5,5′-disubstituted complexes is discussed in comparison with the previously reported [Pt(4,4′-dcbpy)(tdt)]. Electrochemical studies show no major change in the HOMO after movement of the carboxylic acid groups, consistent with assignment of the HOMO as largely dithiolate based. Movement of the carboxylic acid groups makes the diimine electronic character and hence the LUMO of the complexes different. Electrochemical studies show a change to lower energy of the LUMO represented by changes in reduction potential of the compound on moving the carboxylic acid substituents from the 4,4′ to the 5,5′ positions. Both [Pt(5,5′-dcbpy)(tdt)] and [TBA]2[Pt(5,5′-dcbpy)(tdt)] have been used as solar cell sensitisers, with the di-TBA salt giving lower dye loading but superior photovoltaic performance. The consequences of tuning the complex through the position of the carboxylic acid groups are discussed.

Breaking and re-forming the disulfide bond at the high-potential, respiratory-type Rieske [2Fe-2S] center of thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry.

A disulfide bond, adjacent to the [2Fe-2S] cluster, is conserved in all high-potential Rieske proteins from the respiratory and photosynthetic cytochrome bc(1) and b(6)f complexes but is absent from the low-potential, bacterial dioxygenase Rieske proteins. The role of the disulfide is unclear, since cysteine mutants have resulted in only apoprotein. The high stability of the soluble Thermus thermophilus Rieske protein permits chemical reduction of the disulfide bond and characterization of the sulfhydryl (dithiol) form by protein-film voltammetry. The effect of disulfide reduction on the cluster potential is small (DeltaE(0)' <or= -0.04 V) and attributed to relaxation of the disulfide tether between the protein loops ligating the cluster, including possible mechanical strain release and hydrogen-bonding modification. Above pH 6 an additional decrease in potential of the sulfhydryl form is assigned to the nearby negatively charged thiolates (DeltaE(0)' -0.16 to -0.12 V); the histidine-ligand nitrogen pKs are correspondingly increased. Entropies of reduction for the native and dithiolate forms are equal (-48 +/- 5 J K(-1) mol(-1), pH 7-8); thus changes in reduction potential are enthalpic in origin. Following sulfhydryl alkylation the cluster redox properties mirror those of the native protein (DeltaE(0)' approximately -0.1 V) over all pHs. While a sustained electrode potential of -0.85 V fails to reduce the disulfide, the free sulfhydryls recombine upon an oxidative excursion, at low pH, to restore the native redox properties. This unique behavior is attributed to preorganization of the two thiolate groups upon uptake of one or more protons by the sulfhydryl pair.

Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin.

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.

Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature.

The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P. furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles. The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.

Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c

Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential ( E 0′) for each cytochrome, the entropy of reaction, Δ S 0′ Rxn(Δ S 0′ Rxn= S 0′ Red− S 0′ Ox), for the reduction process was determined via the non-isothermal method. Having determined Δ S 0′ Rxn and E 0′, Δ H 0′ was calculated. For rat cytochrome c, it was found that Δ S 0′ Rxn=−43 J mol −1 K −1 for the wild type and −53 J mol −1 K −1 for the Tyr67Phe variant, with the Δ H 0′ for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in Δ S 0′ Rxn. In contrast the measured Δ S 0′ Rxn for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.

Methanol Synthesis from CO2, CO, and H2over Cu(100) and Ni/Cu(100)

The catalytic activity of Cu(100) and Ni/Cu(100) with respect to the methanol synthesis from various mixtures containing CO2, CO, and H2have been studied in a combined UHV/high pressure cell apparatus at reaction conditions,Ptot=1.5 bar andT=543 K. For the clean Cu(100) surface it is found that admission of CO to a reaction mixture containing CO2and H2does not lead to an increase in the rate of methanol formation, which indirectly suggests that the role of CO in the industrial methanol process relates to the change in reduction potential of the synthesis gas. For the Ni/Cu(100) surface it is found that Ni does not promote the rate of methanol formation from mixtures containing CO2and H2. In opposition, admission of CO to the reaction mixture leads to a significant increase in the rate of methanol formation with a turnover frequency/Ni site∼60×the turnover frequency/Cu site at Ni coverages below 0.1 ML making it a rather substantial promoting effect. It is found that the admission of CO to the synthesis gas creates segregation of Ni to the surface, whereas this is not the case for a reaction involving CO2and H2. It is suggested that CO acts strictly as a promotor in the system and we ascribe the increase in activity to a promotion through gas phase induced surface segregation of Ni.

A Novel Series of p−n Diblock Light-Emitting Copolymers Based on Oligothiophenes and 1,4-Bis(oxadiazolyl)-2,5-dialkyloxybenzene

A new series of p−n diblock conjugated copolymers consisting of alternate 1,4-bis(oxadiazolyl)-2,5-dialkyloxybenzene and oligothiophenes with one to three thiophene rings (P1−P3) have been synthesized. The polymers have well-defined structures and exhibit good thermal stability with the onset decomposition temperatures in nitrogen at around 300 °C. The glass transition temperature (Tg) of the polymers decreases with increasing the length of oligothiophene blocks. Both the absorption spectra and photoluminescence spectra shift to longer wavelength with increase in the length of oligothiophene blocks. The emissive color of the polymers could be tuned from blue to green to orange just by increasing the number of thiophene rings in the oilgothiophene blocks from one to three. No obvious change in reduction potential is found for the polymers with variation in the length of oligothiophene blocks. The reduction potential E1/2 of the polymers is measured by cyclic voltammetry to be around −1.7 V vs SCE, comparable to those of poly(cyanoterephthalyidene) (CN-PPV) and other good electron-transporting materials. The oxidation potential of the polymers can be reduced remarkably by increasing the length of oligothiophene blocks. The oxidation potential E1/2 of P3 is measured to be 1.25 V with the onset potential at 1.0 V vs SCE. These values are comparable to those of some hole-injection favorable electroluminescent polymers. The results prove that the p−n diblock structure may be a promising molecular design for synthetically tuning the HOMO and LUMO of conjugated polymers.

Simple dielectric image charge models for electrostatic interactions in metalloproteins

An investigation has been made of the electrostatic potential from an excess point charge near dielectric interfaces by the method of image charges. Two configurations were considered: one is a planar slab of low constant dielectric permittivity enclosed between two infinite regions of larger constant dielectric permittivity, and the other is a dielectric sphere of low constant dielectric permittivity embedded in an infinite region of larger constant dielectric permittivity. The systems are representative of charges near bilayer membranes and globular proteins, respectively. The potentials reduce to simple analytical forms in these configurations. The notion of an ‘effective dielectric permittivity’ εeff is useful, with characteristic distance and orientation dependence caused by the image charge distributions. εeff extends from values between the two constant intrinsic dielectric permittivities, but always larger than the lower of the two, to values which exceed significantly the value in the embedding regions. The spherical model has been compared with observed electrostatic effects in cytochrome c 551, azurin and plastocyanin. The effects refer to reduction potential changes caused by deprotonation, pK a in different oxidation states, charged residue replacement, and the effects of charged inhibitor molecules. Conformational changes are insignificant in these cases. The observable quantities mostly are reproduced well by the simple analytical potential form, pointing to the ready applicability of such models, supplementary to detailed numerical procedures.

Simple dielectric image charge models for electrostatic interactions in metalloproteins

An investigation has been made of the electrostatic potential from an excess point charge near dielectric interfaces by the method of image charges. Two configurations were considered: one is a planar slab of low constant dielectric permittivity enclosed between two infinite regions of larger constant dielectric permittivity, and the other is a dielectric sphere of low constant dielectric permittivity embedded in an infinite region of larger constant dielectric permittivity. The systems are representative of charges near bilayer membranes and globular proteins, respectively. The potentials reduce to simple analytical forms in these configurations. The notion of an ‘effective dielectric permittivity’ εeff is useful, with characteristic distance and orientation dependence caused by the image charge distributions. εeff extends from values between the two constant intrinsic dielectric permittivities, but always larger than the lower of the two, to values which exceed significantly the value in the embedding regions. The spherical model has been compared with observed electrostatic effects in cytochrome c 551, azurin and plastocyanin. The effects refer to reduction potential changes caused by deprotonation, pK a in different oxidation states, charged residue replacement, and the effects of charged inhibitor molecules. Conformational changes are insignificant in these cases. The observable quantities mostly are reproduced well by the simple analytical potential form, pointing to the ready applicability of such models, supplementary to detailed numerical procedures.

Structure-function relationships in Anabaena ferredoxin: correlations between X-ray crystal structures, reduction potentials, and rate constants of electron transfer to ferredoxin:NADP+ reductase for site-specific ferredoxin mutants.

A combination of structural, thermodynamic, and transient kinetic data on wild-type and mutant Anabaena vegetative cell ferredoxins has been used to investigate the nature of the protein-protein interactions leading to electron transfer from reduced ferredoxin to oxidized ferredoxin:NADP+ reductase (FNR). We have determined the reduction potentials of wild-type vegetative ferredoxin, heterocyst ferredoxin, and 12 site-specific mutants at seven surface residues of vegetative ferredoxin, as well as the one- and two-electron reduction potentials of FNR, both alone and in complexes with wild-type and three mutant ferredoxins. X-ray crystallographic structure determinations have been carried out for six of the ferredoxin mutants. None of the mutants showed significant structural changes in the immediate vicinity of the [2Fe-2S] cluster, despite large decreases in electron-transfer reactivity (for E94K and S47A) and sizable increases in reduction potential (80 mV for E94K and 47 mV for S47A). Furthermore, the relatively small changes in Calpha backbone atom positions which were observed in these mutants do not correlate with the kinetic and thermodynamic properties. In sharp contrast to the S47A mutant, S47T retains electron-transfer activity, and its reduction potential is 100 mV more negative than that of the S47A mutant, implicating the importance of the hydrogen bond which exists between the side chain hydroxyl group of S47 and the side chain carboxyl oxygen of E94. Other ferredoxin mutations that alter both reduction potential and electron-transfer reactivity are E94Q, F65A, and F65I, whereas D62K, D68K, Q70K, E94D, and F65Y have reduction potentials and electron-transfer reactivity that are similar to those of wild-type ferredoxin. In electrostatic complexes with recombinant FNR, three of the kinetically impaired ferredoxin mutants, as did wild-type ferredoxin, induced large (approximately 40 mV) positive shifts in the reduction potential of the flavoprotein, thereby making electron transfer thermodynamically feasible. On the basis of these observations, we conclude that nonconservative mutations of three critical residues (S47, F65, and E94) on the surface of ferredoxin have large parallel effects on both the reduction potential and the electron-transfer reactivity of the [2Fe-2S] cluster and that the reduction potential changes are not the principal factor governing electron-transfer reactivity. Rather, the kinetic properties are most likely controlled by the specific orientations of the proteins within the transient electron-transfer complex.

Effect of axial ligand plane reorientation on electronic and electrochemical properties observed in the A67V mutant of rat cytochrome b5.

Mutational studies directed at evaluating the effect of the axial ligand plane orientation on electrochemical properties of cytochrome b5 have been performed. As described in the previous paper, structural consequences of one of these mutations, the A67V mutation, have been evaluated using NMR solution methods. The lack of large shifts relative to the wild-type protein in both the imidazole Ndelta nitrogen and proton resonances of the H63 imidazole ring indicates that the hydrogen bond between the carbonyl of F58 and the imidazole ring of H63 remains intact in this mutant. Effects of the imidazole plane reorientation on the Fe d-orbitals were evaluated on the basis of interpretation of EPR spectra, near-infrared bands associated with ligand-to-metal charge transfer transitions, reorientation of the anisotropy of the paramagnetic center determined by calculation of pseudocontact shifts, and the temperature dependence of the contact-shifted resonances. The dominant effect of the imidazole reorientation appears to have been a destabilization of the d(xz) orbital energy and a reorientation of the d(pi) orbitals. This is surprising in light of the -20 mV shift in the reduction potential of the mutant relative to the wild-type protein and indicates that a destabilization of d(yz)-orbital energy level of the reduced state dictates the observed change in reduction potential. Measured values for the reorganizational energy and heterogeneous electron transfer rates were indistinguishable for wild-type and mutant proteins. This is perhaps surprising, given significant differences in the pattern of electron delocalization into the porphyrin ring observed as significantly altered contact shift patterns. Mutational studies perturbing the H39 imidazole were also performed but with more limited success.

The CXXC motif: a rheostat in the active site.

The active-site CXXC motif of thiol:disulfide oxidoreductases is essential for their catalysis of redox reactions. Changing the XX residues can perturb the reduction potential of the active-site disulfide bond of the Escherichia coli enzymes thioredoxin (Trx; CGPC) and DsbA (CPHC). The reduction potential is correlated with the acidity of the N-terminal cysteine residue of the CXXC motif. As the pKa is lowered, the disulfide bond becomes more easy to reduce. A change in pKa can account fully for a change in reduction potential in well-characterized CXXC motifs of DsbA but not of Trx. Formal analysis of the Nernst equation reveals that reduction potential contains both pH-dependent and pH-independent components. Indeed, the difference between the reduction potentials of wild-type Trx and wild-type DsbA cannot be explained solely by differences in thiol pKa values. Structural data for thiol:disulfide oxidoreductases reveal no single factor that determines the pH-independent component of the reduction potential. In addition, the pH-dependent component is complex when the redox state of the CXXC motif affects the titration of residues other than the thiols. These intricacies enable CXXC motifs to vary widely in their capacity to assist electron flow, and thereby engender a family of thiol:disulfide oxidoreductases that play diverse roles in biochemistry.