Furiosus Rubredoxin Research Articles

Development of Nonheme {FeNO}7 Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron-sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration.

Cluster-Dependent Charge-Transfer Dynamics in Iron-Sulfur Proteins.

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.

Molecular dynamics simulations of metalloproteins: A folding study of rubredoxin from <em>Pyrococcus furiosus</em>

The constant increase of computational power has made feasible to investigate the folding mechanism of small proteins using molecular dynamics (MD). Metal-binding proteins (metalloproteins) are usually complicated to model, largely due to the presence of the metal cofactor. Thus, the study of metal-coupled folding is still challenging. In this work, we addressed the folding process of Pyrococcus furiosus rubredoxin (PfRd), a 53-residue protein binding a single iron ion, using different MD methods. Starting from an extended conformation of the polypeptide chain where we preserved the coordination of the metal ion, a classical MD simulation and an extensive accelerated MD run were performed to reconstruct the folding process of the metal-bound protein. For comparison, we simulated also the dynamics of folded PfRd devoid of the metal cofactor (apo-form), starting from the folded structure. For these MD trajectories, we computed various structural and biochemical properties. In addition, we took advantage of available experimental data to quantify the degree to which our simulations sampled conformations close to the native folded state. We observed that the compaction of the hydrophobic core is the main feature driving the folding of the structure. However, we could not reach a fully folded conformation within our trajectories, because of the incomplete removal of the solvent from the core. Altogether, the various MD simulations, including that of the folded apo-form of the protein, suggest that an improvement in the accuracy of the protein force-field is still needed.

Hyperthermophile Protein Behavior: Partially-Structured Conformations of Pyrococcus furiosus Rubredoxin Monomers Generated through Forced Cold-Denaturation and Refolding

Some years ago, we showed that thermo-chemically denatured, partially-unfolded forms of Pyrococcus furiosus triosephosphateisomerase (PfuTIM) display cold-denaturation upon cooling, and heat-renaturation upon reheating, in proportion with the extent of initial partial unfolding achieved. This was the first time that cold-denaturation was demonstrated for a hyperthermophile protein, following unlocking of surface salt bridges. Here, we describe the behavior of another hyperthermophile protein, the small, monomeric, 53 residues-long rubredoxin from Pyrococcus furiosus (PfRd), which is one of the most thermostable proteins known to man. Like PfuTIM, PfRd too displays cold-denaturation after initial thermo-chemical perturbation, however, with two differences: (i) PfRd requires considerably higher temperatures as well as higher concentrations of guanidium hydrochloride (Gdm.HCl) than PfuTIM; (ii) PfRd's cold-denaturation behavior during cooling after thermo-chemical perturbation is incompletely reversible, unlike PfuTIM's, which was clearly reversible (from each different conformation generated). Differential cold-denaturation treatments allow PfRd to access multiple partially-unfolded states, each of which is clearly highly kinetically-stable. We refer to these as ‘Trishanku’ unfolding intermediates (or TUIs). Fascinatingly, refolding of TUIs through removal of Gdm.HCl generates multiple partially-refolded, monomeric, kinetically-trapped, non-native ‘Trishanku’ refolding intermediates (or TRIs), which differ from each other and from native PfRd and TUIs, in structural content and susceptibility to proteolysis. We find that the occurrence of cold denaturation and observations of TUI and TRI states is contingent on the oxidation status of iron, with redox agents managing to modulate the molecule's behavior upon gaining access to PfRd's iron atom. Mass spectrometric examination provides no evidence of the formation of disulfide bonds, but other experiments suggest that the oxidation status of iron (and its extent of burial) together determine whether or not PfRd shows cold denaturation, and also whether redox agents are able to modulate its behavior.

The Key to the Extraordinary Thermal Stability of P. furiosus Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure

Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.

Assessing the chemical accuracy of protein structures via peptide acidity

Although the protein native state is a Boltzmann conformational ensemble, practical applications often require a representative model from the most populated region of that distribution. The acidity of the backbone amides, as reflected in hydrogen exchange rates, is exquisitely sensitive to the surrounding charge and dielectric volume distribution. For each of four proteins, three independently determined X-ray structures of differing crystallographic resolution were used to predict exchange for the static solvent-exposed amide hydrogens. The average correlation coefficients range from 0.74 for ubiquitin to 0.93 for Pyrococcus furiosus rubredoxin, reflecting the larger range of experimental exchange rates exhibited by the latter protein. The exchange prediction errors modestly correlate with the crystallographic resolution. MODELLER 9v6-derived homology models at ~60% sequence identity (36% identity for chymotrypsin inhibitor CI2) yielded correlation coefficients that are ~0.1 smaller than for the cognate X-ray structures. The most recently deposited NOE-based ubiquitin structure and the original NMR structure of CI2 fail to provide statistically significant predictions of hydrogen exchange. However, the more recent RECOORD refinement study of CI2 yielded predictions comparable to the X-ray and homology model-based analyses.

A Billion-fold Range in Acidity for the Solvent-Exposed Amides of Pyrococcus furiosus Rubredoxin

The exchange rates of the static solvent-accessible amide hydrogens of Pyrococcus furiosus rubredoxin range from near the diffusion-limited rate to a billion-fold slower for the non-hydrogen-bonded Val 38 (eubacterial numbering). Hydrogen exchange directly monitors the kinetic acidity of the peptide nitrogen. Electrostatic solvation free energies were calculated by Poisson-Boltzmann methods for the individual peptide anions that form during the hydroxide-catalyzed exchange reaction to examine how well the predicted thermodynamic acidities match the experimentally determined kinetic acidities. With the exception of the Ile 12 amide, the differential exchange rate constant for each solvent-exposed amide proton that is not hydrogen bonded to a backbone carbonyl can be predicted within a factor of 6 (10 (0.78)) root-mean-square deviation (rmsd) using the CHARMM22 electrostatic parameter set and an internal dielectric value of 3. Under equivalent conditions, the PARSE parameter set yields a larger rmsd value of 1.28 pH units, while the AMBER parm99 parameter set resulted in a considerably poorer correlation. Either increasing the internal dielectric value to 4 or reducing it to a value of 2 significantly degrades the quality of the prediction. Assigning the excess charge of the peptide anion equally between the peptide nitrogen and the carbonyl oxygen also reduces the correlation to the experimental data. These continuum electrostatic calculations were further analyzed to characterize the specific structural elements that appear to be responsible for the wide range of peptide acidities observed for these solvent-exposed amides. The striking heterogeneity in the potential at sites along the protein-solvent interface should prove germane to the ongoing challenge of quantifying the contribution that electrostatic interactions make to the catalytic acceleration achieved by enzymes.

Spatial distribution of dielectric shielding in the interior of Pyrococcus furiosus rubredoxin as sampled in the subnanosecond timeframe by hydrogen exchange

Experimental p K values of ionizable sidechains provide the most direct test for models representing dielectric shielding within the interior of a protein. However, only the strongly shifted p K values are particularly useful for discriminating among models. NMR titration studies have usually found only one or two such shifted p K values in each protein, so that the fitting of the experimental data to a uniform internal dielectric ( ε int) model is not well constrained. The observed variation among proteins for such ε int estimates may reflect nonuniformity of dielectric shielding within each protein interior or qualitative differences between individual proteins. The differential amide kinetic acidities for a series of metal-substituted rubredoxins are shown to be consistent with Poisson–Boltzmann predictions of dielectric shielding that is relatively uniform for all of the amides that are sensitive to the metal charge, a region which corresponds to roughly 1 / 3 of the internal volume. The effective ε int values near 6 that are found in this study are significantly lower than many such estimates derived from sidechain p K measurements. The differing timeframes in which dielectric relaxation can respond to the highly transient peptide anion as compared to the longer lived states of the charged sidechains offers an explanation for the lower apparent dielectric constant deduced from these measurements.

Electrostatic Potential Energy within a Protein Monitored by Metal Charge-Dependent Hydrogen Exchange

Hydrogen exchange measurements on Zn(II)-, Ga(III)-, and Ge(IV)-substituted Pyrococcus furiosus rubredoxin demonstrate that the log ratio of the base-catalyzed rate constants (Δ log k ex) varies inversely with the distance out to at least 12 Å from the metal. This pattern is consistent with the variation of the amide nitrogen pK values with the metal charge-dependent changes in the electrostatic potential. Fifteen monitored amides lie within this range, providing an opportunity to assess the strength of electrostatic interactions simultaneously at numerous positions within the structure. Poisson-Boltzmann calculations predict an optimal effective internal dielectric constant of 6. The largest deviations between the experimentally estimated and the predicted ΔpK values appear to result from the conformationally mobile charged side chains of Lys-7 and Glu-48 and from differential shielding of the peptide units arising from their orientation relative to the metal site.

Crystallization and preliminary X-ray diffraction studies of a hyperthermophilic Rieske protein variant (SDX-triple) with an engineered rubredoxin-like mononuclear iron site

In place of the Rieske [2Fe-2S] cluster, an archetypal mononuclear iron site has rationally been designed into a hyperthermophilic archaeal Rieske [2Fe-2S] protein (sulredoxin) from Sulfolobus tokodaii by three residue replacements with reference to the Pyrococcus furiosus rubredoxin sequence. The resulting sulredoxin variant, SDX-triple (H44I/A45C/H64C), has been purified and crystallized by the hanging-drop vapour-diffusion method using 65%(v/v) 2-methyl-2,4-pentanediol, 0.025 M citric acid and 0.075 M sodium acetate trihydrate pH 4.3. The crystals diffract to 1.63 A resolution and belong to the triclinic space group P1, with unit-cell parameters a = 43.56, b = 76.54, c = 80.28 A, alpha = 88.12, beta = 78.82, gamma = 73.46 degrees. The asymmetric unit contains eight protein molecules.

Role of Native-State Structure in Rubredoxin Native-State Hydrogen Exchange

Amide exchange rates were measured for Pyrococcus furiosus (Pf) rubredoxin substituted with either Zn(II), Ga(III), or Ge(IV). Base-catalyzed exchange rate constants increase up to 3000-fold per unit charge for the highly protected amides surrounding the active site metal, yielding apparent residue-specific conformational energy decreases of more than 8 kcal/mol in a comparison of the Zn(II)- and Ge(IV)-substituted proteins. However, the exchange kinetics for many of the other amides of the protein are insensitive to these metal substitutions. These differential rates are inversely correlated with the distance between the amide nitrogen and the metal in the X-ray structure, out to a distance of at least 12 A, consistent with an electrostatic potential-dependent shifting of the amide nitrogen pK. This strongly correlated distance dependence is consistent with a nativelike structure for the exchange-competent conformations. The electric field potential within the interior of the rubredoxin structure gives rise to a change of as much as a million-fold in the rate for the exchange-competent state of the individual amide hydrogens. Nevertheless, the strength of these electrostatic interactions in Pf rubredoxin appears to be comparable to those previously reported within other proteins. As a result, contrary to the conventional analysis of hydrogen exchange data, for exchange processes that occur via nonglobal transitions, the residual conformational structure will often modulate the observed rates. Although this necessarily complicates the estimation of the conformational equilibria of these exchange-competent states, this dependence on residual structure can provide insight into the conformation of these transient states.

Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: Spatial propagation of differential flexibility in rubredoxin hybrids

The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14-33(Pf)) Cp, that exhibits a 12 degrees increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14-33(Pf)) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14-33(Pf)) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs.

Additivity in Both Thermodynamic Stability and Thermal Transition Temperature for Rubredoxin Chimeras via Hybrid Native Partitioning

Given any operational definition of pairwise interaction, the set of residues that differ between two structurally homologous proteins can be uniquely partitioned into subsets of clusters for which no such interactions occur between clusters. Although hybrid protein sequences that preserve such clustering are consistent with tertiary structures composed of only parental native-like interactions, the stability of such predicted structures will depend upon the physical robustness of the assumed interaction potential. A simple distance cutoff criterion was applied to the most thermostable protein known to predict such a seven-residue cluster in the metal binding site region of Pyrococcus furiosus rubredoxin and a mesophile homolog. Both conformational stability and thermal transition temperature measurements demonstrate that 39% of the differential stability arises from these seven residues.

Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments

Rubredoxins are the simplest type of iron–sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii ( T opt = 85 °C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron–sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron–sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.

Contribution of the multi-turn segment in the reversible thermal stability of hyperthermophile rubredoxin: NMR thermal chemical exchange analysis of sequence hybrids

Pyrococcus furiosus ( Pf) rubredoxin is the most thermostable protein characterized to date. Reflecting the complications arising from irreversible denaturation of this protein, predictions of which structural regions confer differential thermal stability have utilized kinetic stability measurements, hydrogen exchange protection factors, long range hydrogen bond NMR spin couplings, and molecular dynamics simulations, and have primarily implicated the three-stranded β-sheet and the adjacent metal binding site. Herein, NMR chemical exchange experiments demonstrate reversible two-state unfolding at the thermal transition temperature ( T m) for hybrids of Pf and the mesophile Clostridium pasteurianum ( Cp) rubredoxins which interchange residues 14–33, the so-called multi-turn segment. This complementary pair of hybrid rubredoxins exhibits largely additive incremental thermal stabilizations vs. the parental proteins. Both stabilization free energy measurements as well as incremental T m values indicate that a minimum of 37% of the total differential thermal stability resides in this multi-turn segment. Such a proportionality between ΔΔ G and incremental T m values is predicted for hybrid pairs exhibiting thermodynamic additivity in which the differential stability is predominantly enthalpic.

Structural genomics of Pyrococcus furiosus: X-ray crystallography reveals 3D domain swapping in rubrerythrin.

Structural genomics of Pyrococcus furiosus: X-ray crystallography reveals 3D domain swapping in rubrerythrin.

Femtosecond dynamics of rubredoxin: tryptophan solvation and resonance energy transfer in the protein.

We report here studies of tryptophan (Trp) solvation dynamics in water and in the Pyrococcus furiosus rubredoxin protein, including the native and its apo and denatured forms. We also report results on energy transfer from Trp to the iron-sulfur [Fe-S] cluster. Trp fluorescence decay with the onset of solvation dynamics of the chromophore in water was observed with femtosecond resolution ( approximately 160 fs; 65% component), but the emission extended to the picosecond range (1.1 ps; 35% component). In contrast, the decay is much slower in the native rubredoxin; the Trp fluorescence decay extends to 10 ps and longer, reflecting the local rigidity imposed by residues and by the surface water layer. The dynamics of resonance energy transfer from the two Trps to the [Fe-S] cluster in the protein was observed to follow a temporal behavior characterized by a single exponential (15-20 ps) decay. This unusual observation in a protein indicates that the resonance transfer is to an acceptor of a well-defined orientation and separation. From studies of the mutant protein, we show that the two Trp residues have similar energy-transfer rates. The critical distance for transfer (R(0)) was determined, by using the known x-ray data, to be 19.5 A for Trp-36 and 25.2 A for Trp-3, respectively. The orientation factor (kappa(2)) was deduced to be 0.13 for Trp-36, clearly indicating that molecular orientation of chromophores in the protein cannot be isotropic with kappa(2) value of 2/3. These studies of solvation and energy-transfer dynamics, and of the rotational anisotropy, of the wild-type protein, the (W3Y, I23V, L32I) mutant, and the fmetPfRd variant at various pH values reveal a dynamically rigid protein structure, which is probably related to the hyperthermophilicity of the protein.

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.

Rubredoxin Variant Folds without Iron

Pyroccocus furiosus rubredoxin (PFRD), like most studied hyperthermophilic proteins, does not undergo reversible folding. The irreversibility of folding is thought to involve PFRD’s iron-binding site. Here we report a PFRD variant (PFRD-XC4) whose iron binding site was redesigned to eliminate iron binding using a computational design algorithm. PFRD-XC4 folds without iron and exhibits reversible folding with a melting temperature of 82 °C, a thermodynamic stability of 3.2 kcal mol^(-1) at 1 °C, and NMR chemical shifts similar to that of the wild-type protein. This variant should provide a tractable model system for studying the thermodynamic origins of protein hyperthermostability.

Hyperthermophilic redox chemistry: a re-evaluation

The redox chemistry of Pyrococcus furiosus rubredoxin and ferredoxin has been studied as a function of temperature in direct voltammetry and in EPR monitored bulk titrations. The E ms of both proteins, measured with direct voltammetry, have a normal (linear) temperature dependence and show no pH dependence. EPR monitoring is not a reliable method to determine the temperature dependence of the E m: upon rapid freezing the proteins take their conformation corresponding to the freezing point of the solution.