Redox properties of the Fe3+/Fe2+ couple in Arthromyces ramosus class II peroxidase and its cyanide adduct

The thermodynamics of the one-electron reduction of the ferric heme in free and cyanide-bound Arthromyces ramosus peroxidase (ARP), a class II plant peroxidase, were determined through spectro-electrochemical experiments. The data were compared with those for class III horseradish peroxidase C (HRP) and its cyanide adduct, and were interpreted in terms of ligand binding features, electrostatic effects and solvent accessible surface area of the heme group and of catalytically relevant residues in the heme distal site. The values for free and cyanide-bound ARP (−0.183 and −0.390 V, respectively, at 25 °C and pH 7) are higher than those for HRP and HRP-CN. ARP features an enthalpic stabilization of the ferrous state and a remarkably negative reduction entropy, which are both unprecedented for heme peroxidases. Once the compensatory contributions of solvent reorganization are partitioned from the measured reduction enthalpy, the resulting protein-based value for ARP turns out to be less positive than that for HRP by +10 kJ mol⁻¹. The smaller stabilization of the oxidized heme in ARP most probably results from the less pronounced anionic character of the proximal histidine, and the decreased polarity in the heme distal site as compared with HRP, as indicated by the X-ray structures. The surprisingly negative value for ARP is the result of peculiar reduction-induced solvent reorganization effects.     

A simple method for the determination of reduction potentials in heme proteins

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (E_m = −115 mV ± 2 mV, pH 7.0) and human CLOCK (E_m = −111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.     

Evidence for heme release in layer-by-layer assemblies of myoglobin and polystyrenesulfonate on pyrolitic graphite

Layer-by-layer assemblies of myoglobin and polystyrenesulfonate (PSS) on pyrolitic graphite have been investigated with the goal of determining the origin of the voltammetric response of these films. From the similar midpoint potential, coverage and electron transfer behavior compared with those of adsorbed free heme, it was concluded that the observed voltammetric peak is due to heme adsorbed at the electrode surface. This suggests that the interactions between the pyrolitic graphite electrode, PSS and myoglobin can result in heme release from the protein followed by heme adsorption on the electrode.     

Control of reduction potential by protein matrix: lesson from a spherical protein model

 Factors that contribute to the control of reduction potential by protein matrix are examined within a spherical protein model. These include the nonpolar nature of protein matrices, solvent accessibility of the redox center, and net charges and dipoles of surrounding amino acids. Simple rules on their effects are established. In particular, surface charges have little effect on the reduction potential, and polar groups may either increase or decrease the reduction potential, depending on their orientations relative to the redox center. The effects of complex formation, proton titration, and ionic strength are also discussed. Received, accepted: 26 November 1996     

Effects of reduction and ligation of heme iron on the thermal stability of heme-hemopexin complexes

Hemopexin has two homologous domains (N- and C-terminal domains), binds 1 mole of heme per mole with high affinity (K_d < 1 pM) in a low-spin bis-histidyl complex, and acts as a transporter for the heme. Transport is accomplished via endocytosis without degradation of the protein. Factors that affect stability of the heme coordination complex and potentially heme release in vivo were examined. The effects of temperature on hemopexin, its N-terminal domain, and their respective ferri-, ferro-, and CO-ferro-heme complexes were studied using absorbance and circular dichroism (CD) spectroscopy. As monitored with second-derivative absorbance spectra, the higher order structure of apo-hemopexin unfolds with a T_m of 52°C in 50 mM sodium phosphate buffer and is stabilized by 150 mM NaCl (T_m 63°C). Bis-histidyl heme coordination by hemopexin, observed by Soret absorbance, is substantially weakened by reduction of ferri-heme-hemopexin (T_m 55.5°C) to the ferro-heme form (T_m 48°C), and NaCl stabilizes both complexes by 10-15°C. CO binding to ferro-heme-hemopexin restores complex stability (T_m 67°C). Upon cooling, unfolded apo- and ferri-heme-hemopexin extensively refold and recover substantial heme-binding activity, but the characteristic ellipticity of the native protein (UV region) and heme complex (Soret region) are not regained, indicating that altered refolded forms are produced. Lowering the pH from 7.4 to 6.5 has little effect on the stability of the apo-protein but increases the T_m of heme complexes by 5-12°C. The stability of the apo-N-terminal domain (T_m 53°C) is similar to that of intact hemopexin, and the ferri-, ferro-, and CO-ferro-heme complexes of the N-terminal domain have T_m values of 53°C, 33°C, and 75°C, respectively.     

Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwalds approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E° values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.     

The influence of axial ligands on the reduction potential of blue copper proteins

 The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values. We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-S_Met bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials: stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins. The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around the copper site. Received: 7 April 1999 / Accepted: 26 July 1999     

Redox reactivity of the heme Fe<Superscript>3+</Superscript>/Fe<Superscript>2+</Superscript> couple in native myoglobins and mutants with peroxidase-like activity

The reaction enthalpy and entropy for the one-electron reduction of the ferric heme in horse heart and sperm whale aquometmyoglobins (Mb) have been determined exploiting a spectroelectrochemical approach. Also investigated were the T67R, T67K, T67R/S92D and T67R/S92D Mb-H variants (the latter containing a protoheme-l-histidine methyl ester) of sperm whale Mb, which feature peroxidase-like activity. The reduction potential (E°′) in all species consists of an enthalpic term which disfavors Fe³⁺ reduction and a larger entropic contribution which instead selectively stabilizes the reduced form. This behavior differs from that of the heme redox enzymes and electron transport proteins investigated so far. The reduction thermodynamics in the series of sperm whale Mb variants show an almost perfect enthalpy–entropy compensation, indicating that the mutation-induced changes in are dominated by reduction-induced solvent reorganization effects. The modest changes in E°′ originate from the enthalpic effects of the electrostatic interactions of the heme with the engineered charged residues. The small influence that the mutations exert on the reduction potential of myoglobin suggests that the increased peroxidase activity of the variants is not related to changes in the redox reactivity of the heme iron, but are likely related to a more favored substrate orientation within the distal heme cavity.     

Rationalization of the reduction potentials within the series of the high potential iron-sulfur proteins

An analysis of the factors affecting reduction potentials within a series of high potential iron-sulfur proteins (HiPIP) has been performed by calculating the different contributions to the variation of electrostatic energy upon addition of one electron to the oxidized form of the protein. Molecular dynamics calculations were used to generate model structures of HiPIPs for which X-ray data are not available, starting from the known structures of highly homologous proteins. We have calculated and analyzed the contributions to the electrostatic energy deriving from the net charges present on the surface of the protein, the partial charges present on the uncharged residues, the polarizability of the protein atoms, the solvent dipoles and polarizabilities. A positive correlation with the reduction potentials was found only for the contribution due to the net charge of the protein which, in the absence of other factors such as differences in the coordination properties and in reorganizational energy upon reduction, is proposed to represent the determining effect for the large variation in reduction potential within this series of biological electron carriers.     

Isolation and characterization of two soluble heme c-containing proteins from Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum has been shown to possess two previously undetected heme c-containing, soluble proteins. One is an acidic, c-type cytochrome with a molecular weight of 12 300 and an oxidation-reduction midpoint potential (at pH 8.0) of ?82 mV. The other protein is a basic protein with a molecular weight of 11 900 and an oxidation-reduction midpoint potential (at pH 8.0) of ?110 mV. The basic protein, in both oxidized and reduced forms, has optical spectra similar to those of myoglobin and the oxidized C. vinosum protein exhibits a high-spin heme EPR spectrum similar to that of metmyoglobin. Furthermore, the basic C. vinosum protein binds CO and O₂. The spectra of the CO and O₂ complexes show significant similarities with the respective myoglobin complexes. Possible functions for an O₂-binding protein in C. vinosum are discussed.     

An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase

Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters.     

Structural stability and heme binding potential of the truncated human dual oxidase 2 (DUOX2) peroxidase domain

The essential role of human dual oxidase 2 (hDUOX2) in thyroid hormone biosynthesis defines this member of the NOX/DUOX family, whose absence due to mutation has been directly related to disease, specifically hypothyroidism. Both human DUOX isoforms, hDUOX1 and hDUOX2, are expressed in thyroid tissue; however, hDUOX1 cannot compensate for inactivation of hDUOX2, suggesting that each enzyme is differentially regulated and/or functions in a unique manner. In efforts to uncover relevant structural and functional differences we have expressed and purified the peroxidase domain of hDUOX2_1–599 for direct comparison with the previously studied hDUOX1_1–593. As was shown for hDUOX1, the truncated hDUOX2 domain purifies without a bound heme co-factor and displays no peroxidase activity. However, hDUOX2_1–599 displays greater stability than hDUOX1_1–593. Surprisingly, upon titration with heme, both isoforms bind heme with a low micromolar affinity, demonstrating that they retain a heme binding site. A conformational difference in the full-length protein and/or a protein–protein interaction may be required to increase the heme binding affinity.     

Energetics of the one-electron reduction steps of riboflavin,FMN and FAD to their fully reduced forms

The one-electron reduction potentials (E¹₇) of riboflavin, FMN and FAD have been determined using pulse radiolysis from the position of the electron-transfer equilibria between flavins and reference quinones in aqueous solution. The average value for all three flavins E¹₇(F/FH^.) = ? 314 ± 8 mV is used to calculate the second-electron reduction potential of the flavins E²₇(FH^./FH₂(FH^?)) = ? 124 mV.     

Evaluations of reduction potential data in relation to coupling, kinetics and function

 This commentary addresses the effect and measurement of time-dependent contributions to reduction potentials. Reduction potentials form the basis for many quantitative or semi-quantitative judgements in biological redox chemistry. However, since data are obtained under an assumption of equilibrium being established, their relevance to biological functions requires consideration of the kinetics of the subprocesses that contribute to or influence the overall free energy change. Initial and final states effective in rapid and complex biological functions may differ considerably from those analysed after slow equilibration in a sample tube. A shortcoming of traditional potentiometric measurements is that the time domain is not probed. Voltammetry, a technique that has been much less widely applied in biological chemistry than in chemistry, examines redox transformations in both potential and time domains, and may enable a more realistic picture to be derived. Received, accepted: 26 November 1996     

Direct electrochemistry and electrocatalysis of heme proteins immobilized on gold nanoparticles stabilized by chitosan

Three heme proteins, myoglobin, hemoglobin, and cytochrome c, have been adsorbed onto chitosan-stabilized gold nanoparticles (Chit-Aus) modified Au electrode via a molecule bridge like cysteine. UV-vis spectra indicated that the proteins on Chit-Aus films retained near-native secondary structures. The fabricated procedures and electrochemical behaviors of proteins on such an interface were characterized with electrochemical impedance spectra and cyclic voltammetric techniques. It was demonstrated that Chit-Aus film could not only offer a friendly environment to immobilize protein molecules but also enhance the electron transfer ability between protein molecules and underlying electrode. The effects of scan rate and pH on the electrochemical behaviors of each heme protein are discussed in detail. The resultant electrode displayed an excellent electrocatalytic response to the reduction of H(2)O(2), long-term stability, and good reproducibility.     

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD₅₀ = 40 μM tellurite, 40 min exposure). The organism reduced 70% of 40 μM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF‐Ts, and PPIase) displayed increased abundance in tellurite‐stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3‐hydroxy acyl‐CoA dehydrogenase, acyl‐CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.     

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c

 The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50  °C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E°′ observed with increasing ionic strength [ΔE°′_IS =(E°′) _{I =0.1 M}–(E°′) _{I =0 M}=–0.035 V at 25  °C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factorized out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK _a for a residue deprotonation is the key event of this conformational change. Received: 7 April 1999 / Accepted: 19 July 1999     

The effect of ambient redox potential on the transient electron spin echo signals observed in chloroplasts and photosynthetic algae

Time-resolved electron spin echo (ESE) studies were carried out at room temperature on chloroplast preparations and whole cells of photosynthetic algae. The signals observed exhibit the unexpected special ESE signal which we have proposed to be the result of transient interactions between P⁺-700 and an early electron acceptor of Photosystem I (Thurnauer, M.C. and Norris, J.R. (1980) Chem. Phys. Lett. 76, 557–561). The intensity of the special ESE signal decreases with the chemical reduction of the Center A-Center B complex. The results suggest that in the untreated photosynthetic systems we are initially observing P⁺-700 as it interacts with the reduced acceptor which precedes the Center A-Center B complex. Then the decay of the special ESE signal (approx. 170 ns) gives the lifetime of this reduced acceptor as it participates in forward electron transport.