Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

A regulatory invertase from sugar cane leaf-sheaths

A soluble β-fructofuranosidase was isolated from sugar cane leaf-sheaths. The enzyme attacks sucrose with an activation energy of 5700 cal/mol above 30° and 17 000 cal/mol below 30°. The enzyme was inhibited by the reaction products. Glucose is a simple non-competitive inhibitor, but fructose is a competitive inhibitor. Kinetic studies using double reciprocal plots and replots of 1/K_i, slope vs inhibitor concentration showed that fructose binds to two interacting sites of the enzyme. Per cent residual activity plotted against inhibitor concentration, and Hill plots confirmed the regulatory properties of the invertase. n was found to be close to 2, the number of binding sites established with the double reciprocal method. The tissue and cellular levels of sucrose, fructose and glucose were measured. Fructose was found at inhibitory concentrations confirming that the activity of the enzyme is probably modulated by the hexose pool of the leaf-sheaths.     

Energetics of formation of electron-transfer complexes between asymmetric species

The energetics of formation of diastereomeric electron-transfer complexes between L-dopa or L-adrenaline and iron(III) complex ions bound to poly(L-glutamate) or poly(D-glutamate) were measured by microcalorimetry at 25°C and pH 7. When the association of substrates by Fe-polypeptide systems was virtualy complete, ΔH_ll = 1.3 ± 0.1 and ΔH_dl = 0.9 ± 0.1 kcal/mol with both substrates. A diastereomeric discrimination energy of 400 ± 100 cal/mol is thus observed, which compares satisfactorily with that directely obtained by differential microcalorimetric measurements. These results are fully consistent with previous findings indicating that thermodynamic effects are of minor importance in the overall stereoselectivity of the electron transfer reactions investigated.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca²⁺. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) of 105 ± 15 kcal/mol, in the absence of Ca²⁺. In the presence of Ca²⁺ concentrations sufficient to saturate its sites on Factor IX, the T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca²⁺, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca²⁺, Factor IXα displayed thermograms characterized by a T_M of 51.0 ± 0.5 °C and a ΔH_c of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca²⁺ (the values in the presence of saturating Ca²⁺ are given in parentheses), undergoes thermal denaturation with a T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca²⁺ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.     

A study on intra-particle diffusion effects in enzymatic reactions: glucose-fructose isomerization

In this work different aspects of the glucose-fructose enzymatic isomerization, using immobilized glucose isomerase, are studied and quantified. Reaction temperatures range from 40?°C to 60?°C. Intra-particle effective diffusivities (D _e), determined after uptake experiments, are between 1.20?×?10^?6?cm²/s, at 40?°C, and 2.52?×?10^?6?cm²/s, at 60?°C. The estimated energy of activation for diffusion (E _aD) is 7.71?kcal/mol. No significant adsorption of the sugars on the support gel matrix is observed. Crushed particles (φ = 150–350?μ) are used during kinetic experiments. For this range of particle diameters, inherent kinetics is approached. A reversible Michaelis–Menten rate equation is fitted to the data, providing the following parameters at pH = 7.0: k ₀ = 2.15?×?10^?6?g/IU/s; E _a/R = 8998?K. Glucose (K _G) and fructose (K _F) affinity constants are essentially the same, ranging from 0.190?M, at 40?°C to 0.305?M, at 60?°C. The thermodynamic equilibrium constant is determined for the three temperatures, and the heat of reaction, estimated from a Van't Hoff plot, is ΔH = 1682?cal/mol. Independent experiments, where the reaction occurs in the presence of significant intra-particle mass transfer resistance, are used as validation tests.     

Reversible thermal unfolding of thermostable phosphoglycerate kinase. Thermostability associated with mean zero enthalpy change.

Reversible thermal denaturation of phosphoglycerate kinases (E.C. 2.7.2.3) from an extremely thermophilic bacterium Thermus thermophilus and from yeast were studied by measuring their circular dichroism and fluorescence intensity. The thermal denaturation in the presence of guanidine hydrochloride was completely reversible. The thermodynamic parameters for the reaction were calculated based on a two-state mechanism. The free energy changes in denaturation at 25 °C in the absence of denaturant were estimated to be 11.87 ± 0.21 kcal/mol for T. thermophilus phosphoglycerate kinase and 5.33 ± 0.13 kcal/mol for that of yeast. It was found that the van't Hoff plot of the equilibrium constant for the denaturation reaction was almost independent of temperature in the temperature range 0 to 60 °C for T. thermophilus phosphoglycerate kinase, while that of yeast phosphoglycerate kinase was strongly temperature-dependent as reported for other thermolabile proteins. The enthalpy change in denaturation varies from 0.03 to 6.2 kcal/mol (0 to 60 °C) for T. thermophilus phosphoglycerate kinase and from ?27 to 31 kcal/mol (10 to 35 °C) for yeast enzyme. The entropy change in denaturation varies from ?3.9 to 21 entropy units for T. thermophilus phosphoglycerate kinase and ?96 to 104 entropys unit (10 to 35 °C) for yeast enzyme. The heat capacity change in denaturation is between 1.4 and 63 cal/deg. mol for the thermophile enzyme and between 1530 and 1750 cal/deg. mol for yeast enzyme at 20 °C. The observations that the enthalpy changes as well as the heat capacity changes in denaturation of the thermophilic enzyme were negligibly small suggest an explanation for the unusual stability to heat of T. thermophilus phosphoglycerate kinase.We also propose three possible mechanisms for the thermostability of proteins in general.     

Heat stabilization dependence on redox state of cytochrome cd1 oxidase from Pseudomonas aeruginosa

The irreversible thermal denaturation of cytochrome cd₁ oxidase from $\text{P.}\text{aeruginosa}$ as a function of the oxidation-reduction states of its hemes was observed with a differential scanning calorimeter. Upon full reduction of the four hemes, the apparent denaturation temperature decreases by about 10° and the denaturation enthalpy decreases slightly: oxidized, 5.9 cal/gm; reduced, 5.4 cal/gm. At pH 7.5, the first order rate constants for denaturation at 90°C are: reduced, 33 × 10^?3s^?1; oxidized, 3 × 10^?3s^?1. Thus, oxidation of the hemes reuults in heat stabilization of the cytochrome oxidase. The activation energy for denaturation of fully reduced oxidase, 53 kcal/mol, is less than that for fully oxidized protein (73 kcal/mol).     

Reversible thermal unfolding of thermostable cytochrome c-552.

Reversible thermal denaturation of cytochrome c-552 from the extremely thermophilic bacterium Thermus thermophilus was studied by circular dichroism and fluorescence spectroscopy. Thermal denaturation in the presence of guanidine hydrochloride is completely reversible. The thermodynamic parameters for the reaction have been calculated based on a two-state mechanism. The free energy change on denaturation (ΔG) at 25 °C in the absence of denaturant is estimated to be 28.5 ± 0.15 kcal/mol, which is larger than that of cytochrome c from mesophilic organisms. The temperature of maximum stability is approximately 27 °C, which is higher than those of cytochromes c from mesophilic organisms (9 to 12 °C). The temperature dependences of the enthalpy and entropy changes are similar to those of cytochromes c from mesophilic organisms. The heat capacity change on denaturation is between 1250 and 1680 cal/deg mole, which is similar to those of cytochromes c from mesophilic organisms (1500 to 2500 cal/deg mol). From these results, it has been concluded that T. thermophilus cytochrome c is more stable than cytochromes c from mesophilic organisms by virtue of the fact that the free energy change for denaturation is greater and has its maximum at a higher temperature.     

Calorimetric measurement of enthalpy change in the isothermal helix-coil transition of poly(L-ornithine) in aqueous solution.

The enthalpy change associated with the isothermal pH-induced uncharged coil-to-helix transition ΔH_h° in poly(L -ornithine) in 0.1 N KCl has been determnined calorimetrically to be ?1530 ± 210 and ?1270 ± 530 cal/mol at 10° and 25°C, respectively. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH_h°, the observed calorimetric heat was corrected for the heat of breaking the sample cell, the heat of dilution of HCl, the heat of neutralization of the OH^? ion, and the heat of ionization of the δ-amino group in the random coil. The latter was obtained from similar calorimetric measurements on poly(D ,L -ornithine). Since it was discovered that poly(L -ornithine) undergoes chain cleavage at high pH, the calorimetric measurements were carried out under conditions where no degradation occurred. From the thermally induced uncharged helix–coil transition curve for poly(L -ornithine) at pH 11.68 in 0.1 N KCl in the 0°–40°C region, the transition temperature T_tr and the quantity (?θ_h/?T)_Ttr have been obtained. From these values, together with the measured values of ΔH_h°, the changes in the standard free energy ΔG_h° and entropy ΔG_h°, associated with the uncharged coil-to-helix transition at 10°C have been calculated to be ?33 cal/mol and ?5.3 cal/mol deg, respectively. The value of the Zimm–Bragg helix–coil stability constant σ has been calculated to be 1.4 × 10^?2 and the value of s calculated to be 1.06 at 10°C, and between 0.60 and 0.92 at 25°C.     

The effect of temperature on the self-association of S5 and on the association of S5 with S8 as determined by sedimentation equilibrium

Solutions of proteins S5 and S8 from the Escherichia coli 30 S ribosomal subunit have been examined by sedimentation equilibrium methods as a function of temperature for their behavior in solution as isolated components and in mixtures. The standard enthalpy and entropy at 4 °C for the isodesmic self-association of S5 were determined from a study over the temperature range of 3 to 33 °C to be 0.1 ± 0.9 kcal/mol and 18 ± 3 cal/(mol × deg), respectively. The protein S8 remained monomeric over the same range of temperature. The standard enthalpy and entropy at 4 °C for the association of S5 and S8 were determined on mixtures from a study over the temperature range of 3 to 27 °C to be ?0.4 ± 1.6 kcal/mol and 20 ± 6 cal/(mol × deg), respectively. Based on these values and the previously determined standard Gibbs free energies (S. H. Tindall and K. C. Aune, 1981, Biochemistry20, 4861–4866), the driving force for the self-association of S5 and the association of S5 with S8 could be interpreted as being derived from the expulsion of water upon ion pair formation at the interaction sites.     

Purification of endo- and exolaminarinase and partial characterization of the exoacting form from the copepod acartia clausi (Giesbrecht, 1889)

• 1.1. The copepod Acartia clausi exhibited two laminarinases (exo- and endo-acting forms) purified by gel chromatography followed by affinity chromatography. Specific antibodies have been raised against the purified exolaminarinase antigen.
• 2.2. A single band of protein appeared on a polyacrylamide disc gel electrophoresis; its mol. wt is 21,000.
• 3.3. Biochemical properties of the purified enzyme showed a maximum activity at pH 5.2 and a temperature of 40°C with laminarin as substrate. The thermal stability of the enzyme and the effect of various cations on its activity were examined. The enzyme hydrolyses specifically the β(1–3) linked polysaccharides and had no activity against the α(1–4) or β(1–4) disaccharides or polysaccharides.
• 4.4. The kinetic parameters V_m and K_m vary with the temperature; the affinity constant (K_a) was maximum between 25–30°C. The Arrhenius plot defined two values of energy of activation: 7980 cal/mole and 17,506 cal/mole.
• 5.5. From the purification scheme the exoacting form appears to be largely dominant over the endoacting form.

     

Interactive effects of low atmospheric CO2 and elevated temperature on growth, photosynthesis and respiration in Phaseolus vulgaris

For most of the past 250 000 years, atmospheric CO₂ has been 30–50% lower than the current level of 360 μmol CO₂ mol^–1 air. Although the effects of CO₂ on plant performance are well recognized, the effects of low CO₂ in combination with abiotic stress remain poorly understood. In this study, a growth chamber experiment using a two-by-two factorial design of CO₂ (380 μmol mol^–1, 200 μmol mol^–1) and temperature (25/20 °C day/night, 36/29 °C) was conducted to evaluate the interactive effects of CO₂ and temperature variation on growth, tissue chemistry and leaf gas exchange of Phaseolus vulgaris. Relative to plants grown at 380 μmol mol^–1 and 25/20 °C, whole plant biomass was 36% less at 380 μmol mol^–1× 36/29 °C, and 37% less at 200 μmol mol^–1× 25/20 °C. Most significantly, growth at 200 μmol mol^–1× 36/29 °C resulted in 77% less biomass relative to plants grown at 380 μmol mol^–1× 25/20 °C. The net CO₂ assimilation rate of leaves grown in 200 μmol mol^–1× 25/20 °C was 40% lower than in leaves from 380 μmol mol^–1× 25/20 °C, but similar to leaves in 200 μmol mol^–1× 36/29 °C. The leaves produced in low CO₂ and high temperature respired at a rate that was double that of leaves from the 380μmol mol^–1× 25/20 °C treatment. Despite this, there was little evidence that leaves at low CO₂ and high temperature were carbohydrate deficient, because soluble sugars, starch and total non-structural carbohydrates of leaves from the 200μmol mol^–1× 36/29 °C treatment were not significantly different in leaves from the 380μmol mol^–1× 25/20 °C treatment. Similarly, there was no significant difference in percentage root carbon, leaf chlorophyll and leaf/root nitrogen between the low CO₂× high temperature treatment and ambient CO₂ controls. Decreased plant growth was correlated with neither leaf gas exchange nor tissue chemistry. Rather, leaf and root growth were the most affected responses, declining in equivalent proportions as total biomass production. Because of this close association, the mechanisms controlling leaf and root growth appear to have the greatest control over the response to heat stress and CO₂ reduction in P. vulgaris.     

The purification and properties of a thiaminase I enzyme from nardoo (Marsilea drummondii)

Fronds of the fern nardoo (Marsilea drummondii) contain a thiaminase I enzyme at very high levels of activity. Highest levels of enzyme activity were found in vigorously growing plant material. The thiaminase I has been purified to a final sp act value of 2.07 μkat/mg protein at 30° and pH 6.5. It was shown to have similar properties to thiaminase I enzymes purified from bracken fern, rock fern and freshwater mussels. These enzymes have MW values in the range 93 000–115 000, energies of activation of 14 000 cal mol, pH optima of 8–9 and are quite stable in the pH range 3 to 12 and to extended incubation at 55°. The temperature for 50 % denaturation is 60–65°. p-CMB, mersalyl acid and HgCl₂ (10^t-6 M) are potent inhibitors, but monoiodacetic acid (10^?4 M) has no effect. A wide range of heterocyclic bases, sulphydryl compounds, and amines, including the non-aromatic amines 6-aminohexanoic acid and ethanolamine, act as co-substrates in the thiaminase I reaction; however, their effectiveness is dependent on both their degrees of basicity and to some extent, their stereochemistry. When the co-substrate activity of a range of substituted anilines were compared, no correlation was found between the degree to which the base activates the reaction and the pK_b (or Hammett's sigma constant) of the base.     

The random coil → β transition of copolymers of L-lysine and L-isoleucine: Potentiometric titration and circular dichroism studies

Copolymers of L -lysine and L -isoleucine [poly(L -Lys^f,L -Val^{1 ? f})] containing 4–15% isoleucine were investigated using potentiometric titration and circular dichroism (CD) spectroscopy. With increasing isoleucine content, β-sheet formation is favored over α-helix formation at high pH and room temperature. The fraction of β-sheet present, as a function of pH, calculated from titrations of poly(L -Lys^85.2,L -Ile^14.8), agreed well with data obtained from CD studies for the same copolymer. Thermodynamic parameters were determined from titrations using the method of Zimm and Rice; the partial free energy (ΔG°_{C → β}) at 25° for the coil-to-β-sheet transition for isoleucine was estimated to be ?515 cal/mol; from the temperature dependence of free energy, the partial entropy (ΔS°_cβ), and the partial free enthalpy (ΔH°_{c → β}) of the coil → β transition for isoleucine is estimated to be 2.6 e.u. and 260 cal/mol, respectively. The partial thermodynamic parameters obtained for lysine are in good agreement with literature values. It is concluded from these studies that isoleucine has a very high potential for a β-sheet formation.     

High sensitivity differential scanning calorimetry of the bilayer to hexagonal phase transitions of diacylphosphatidylethanolamines

The bilayer to hexagonal phase transition of dioleoylphosphatidylethanolamine has been detected for the first time by differential scanning calorimetry. The observed transition is dependent on scan rate. This dependence can be explained by assuming that at rapid scan rates, the rate of conversion of bilayer to hexagonal phase is too slow at low temperatures for equilibration to take place. At higher temperatures the rate of interconversion becomes more rapid. The transition is observed to occur at 14°C using a scan rate of 0.74 K/min while it is centered at 8°C using a scan rate of 0.19 K/min. The enthalpy of the transition is 290 ± 40 cal/mol lipid and the transition is characterized by a ΔCp of −9 ± 1 mcal K⁻¹ (g lipid)⁻¹. The bilayer to hexagonal phase transition of dielaidoylphosphatidylethanolamine and of 1-palmitoy1-2-oleoylphosphatidylethanolamine occurs at 65.6°C and 71.4°C, respecitvely, with a corresponding transition enthalpy of 450 ± 20 and 400 ± 30 cal/mol lipid. The transitions of these phosphatidylethanolamines, occuring at higher temperatures, are independent of scan rate and show a higher degree of cooperativity than that of dioleoylphosphatidylethanolamine. Compared with the gel to liquid-crystalline transition of bilayer phospholipids the transition to hexagonal phase has a much lower enthalpy.     

Application of ab initio molecular-orbital calculations to the structural moieties of carbohydrates. Part VI

Ab initio RHF/4–31G molecular-orbital calculations have been conducted on methoxymethyl formate and methoxymethyl acetate as models for examining the anomeric effect and stereochemistry of 1-O-acetylglycopyranoses. The results indicate that, as with the methyl glycopyranosides, the α-⁴C₁(D) configurations are more stable than the β-⁴C₁(D), except that the energy difference is more dependent on the disposition about the glycosidic bond. The lowest-energy conformations occur with glycosidic torsion-angles of ?  180°, where the anomeric energy is about 4 kcal/mol. There is a secondary energy-minimum at ?  90°, for which the anomeric energy is less, about 2 kcal/mol. This orientation corresponds to the conformation most commonly observed in the crystal structures of peracetylated glycopyranoses. Small differences in the CO single-bond lengths, which are observed experimentally in both the α and β anomers, are reproduced by the theoretical calculations.     

The effect of pH and temperature on the kinetics of native and altered glycogen phosphorylase.

Data on the effect of pH and temperature on the kinetics of rabbit muscle phosphorylases a and b and reduced phosphorylase b (α-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) with glycogen as the saturating and inorganic phosphate the variable substrate are presented. The kinetic profiles as a function of pH are similar for these enzyme species except that the positions of the pH-maximal velocity profiles for reduced phosphorylase b are relatively invariant in the 15 °–30 ° range, whereas the “native” phosphorylases exhibit a substantial shift of the lower pH limb of the profile toward the acid side when the temperature is lowered from 30 to 15 °C. It is proposed that a group with a pK near 6.0 at 30 °C determines the acid limb of maximal velocity profiles. The phosphoryl moiety of enzyme bound pyridoxal 5′-phosphate is suggested for this group. A conformational transition in the protein, which is somehow modified when the aldimine bond between protein and pyridoxal 5′-phosphate is reduced, is invoked to account for the large decrease of this acid side apparent pK for the ternary complex of native phosphorylases when the temperature is lowered. A group with a pK near 7.1 and a heat of ionization of about 8000 cal/mol determines the alkaline limb of maximal velocity profiles at 30 °C. An imidazoyl ring ionization of an enzyme histidyl group is proposed to account for this behavior. In the enzyme-glycogen binary complex, the apparent heat of ionization of this group has an anomalous value of about ?10,000 cal/ mol. It is suggested that a neighboring amino or arginyl guanidinium group is able to interact with the imidazoyl ring in the absence of bound inorganic phosphate to cause this anomalous behavior. The effect of pH on K_m for inorganic phosphate is simply explained by a group with a pK of 6.56 and low heat of ionization. The data are interpreted to indicate that the dianion of inorganic phosphate is the true substrate for all forms of phosphorylase. The kinetic results of this report are closely compared with other kinetic data in the literature on mammalian, plant, and bacterial α-glucan phosphorylases and general overall similarity is demonstrated. Various methods for analyzing pH-kinetic data for enzymes are briefly discussed, and the crucial difference in conclusions the choice of method can make is demonstrated with our data.     

Conformation of adenosine 3′,5′-monophosphate in solution as studied by the NMR-desert method II. Self-association and temperature-dependent glycosidic isomerization at pH 7

A study has been made of the association and the temperature-dependent conformation of adenosine 3′,5′-monophosphate (cyclic AMP) in a neutral aqueous (²H₂O) solution by means of proton magnetic resonance chemical shift and relaxation. The concentration and temperature-dependent chemical shifts of H(1′), H(2), and H(8), have enabled us to estimate the self-association constant, K_a = 1.1 ± 0.3 M^?1 at 25°C and thermodynamic parameters ΔH = ?5.8 ± 1.5 kcal/mol and ΔS (25°C) = ?19.0 ± 3 cal/mol per degree.The NMR-DESERT (Deuterium Substitution Effect on Relaxation Times) method has been utilized for the determination of the syn-anti conformational equilibrium in the monomeric state and for the determination of the mutual orientation of the two adenine rings in the dimeric state of cyclic AMP. The molecules were found to coexist with nearly equimolarity of syn-anti conformers and thermal activation of the molecules perturbs the syn-anti conformational equilibrium to comprise the syn form in preference at higher temperature. The glycosidic isomerization (from anti to syn) was found to be characterized both by a positive enthalpy change and by a positive entropy change. The cyclic AMP molecules prefer to take a ‘trans-stacking’ conformation in the dimeric state where the two molecules are arranged in such a way that the H(2) of one molecule is close to the H(8) of the other.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.