Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

 The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N₂S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential. Received: 20 December 1996 / Accepted: 26 March 1997     

Thermodynamics of the alkaline transition of cytochrome c.

The apparent equilibrium constant (Kapp) of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pKapp values is biphasic and yields two distinct sets of DeltaH degrees 'AT and DeltaS degrees 'AT values below and above approximately 40 degrees C. In the low-temperature range, the process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss. The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuzzi, G., Borsari, M., Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temperature ranges. Extrapolation of the Kapp values at null ionic strength allowed the determination of the thermodynamic equilibrium constants (Ka) at each temperature, hence of the "true" standard thermodynamic parameters of the transition. The pKa value at 25 degrees C was found to be 8.0. A pKapp value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Hückel theory fits the ionic strength dependence of the pKapp values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential of this protein class. It is apparent that the increase in the pKapp values with increasing ionic strength is for the most part an entropic effect.     

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.     

Control of metalloprotein reduction potential: compensation phenomena in the reduction thermodynamics of blue copper proteins

The reduction thermodynamics (Delta H degrees '(rc) and Delta S degrees '(rc)) for native Paracoccus versutus amicyanin, for Alcaligenes faecalis S-6 pseudoazurin, and for the G45P, M64E, and K27C variants of Pseudomonas aeruginosa azurin were measured electrochemically. Comparison with the data available for other native and mutated blue copper proteins indicates that the features of metal coordination and the electrostatic potential due to the protein matrix and the solvent control the reduction enthalpy in a straightforward way. However, the effects on the reduction potential are rather unpredictable owing to the entropic contribution to E degrees ', which is mainly determined by solvent reorganization effects. Analysis of all the Delta H degrees '(rc) and Delta S degrees '(rc) values available for this protein class indicates that enthalpy-entropy compensation occurs in the reduction thermodynamics of wt cupredoxins from different sources, as well as for mutants of the same species. The findings indicate that the reduction enthalpies and entropies for these species are strongly affected by reduction-induced reorganization of solvent molecules within the solvation sphere of the protein. The absence of a perfect enthalpy-entropy compensation is due to the fact that while the differences between reduction entropies are dominated by solvent reorganization effects, those between reduction enthalpies are significantly controlled by intrinsic molecular factors related to the selective stabilization of the reduced form by coordination features of the copper site and electrostatic effects at the interface with the protein matrix.     

Redox properties and acid-base equilibria of zucchini mavicyanin

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.     

Perioperative Myocardial Injury after Adult Heart Transplant: Determinants and Prognostic Value

MethodsData on 362 consecutive recipients (mean age: 47.8±13.7, 20.2% female, 18.2% diabetics, 22.1% with previous cardiac operations, 27.6% hospitalized, 84.9±29.4 ml/min preoperative glomerular filtration rate) were analyzed using multivariable logistic regression modeling. Target outcomes were determinants of troponin release, early graft failure (EGF), acute kidney injury (AKI) and operative death.ResultsMean cTnI release measured 24 hours after transplant was 10.9±11.6 μg/L. Overall hospital mortality was 10.8%, EGF 10.5%, and AKI was 12.2%. cTnI release>10 μg/L proved an independent predictor of EGF (OR 2.2; 95% CI, 1.06–4.6) and AKI (OR 1.031; 95% CI, 1.001-1.064). EGF, in turn, proved a determinant of hospital mortality. Risk factors for cTnI>10 μg/L release were: status 2B (OR 0.35; 95% CI, 0.18-0.69, protective), duration of the ischemic period (OR 1.006; 95% CI, 1.001-1.011), previous cardiac operation (OR 2.9; 95% CI, 1.67-5.0), and left ventricular hypertrophy (OR 3.3; 95% CI, 1.9-5.6).ConclusionsMyocardial enzyme leakage clearly emerged as an epiphenomenon of more complicated clinical course. The complex interplay between surgical procedure features, graft characteristics and recipient end-organ function highlights cTnI release as a risk marker of graft failure and acute kidney injury. The search for optimal myocardial preservation is still an issue.     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

Multidomain Human Peroxidasin 1 Is a Highly Glycosylated and Stable Homotrimeric High Spin Ferric Peroxidase

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of −233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (k_cat/K_M^app) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.     

Cloning,expression, and physicochemical characterization of a new diheme cytochrome <Emphasis Type="Italic">c</Emphasis> from <Emphasis Type="Italic">Shewanella baltica</Emphasis> OS155

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV–vis, magnetic circular dichroism, and ¹H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid–base equilibria with pK _a values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of −0.144 and −0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye–Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°′.     

Ligand loop effects on the free energy change of redox and pH-dependent equilibria in cupredoxins probed on amicyanin variants

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic "memory" of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.