Mechanism of the reaction of hydrated electrons with ferrocytochrome c.

1. The hydrated electron reacts with ferrocytochrome c to form an unstable intermediate. This intermediate decays in a first-order manner to give, in the first instance, a product which has a similar absorption spectrum in the range 400-610 nm as normal ferricytochrome c. 2. At 21 degrees C the rate constant for the reaction of hydrated electrons with ferrocytochrome c at pH 7.4 (2 mM phosphate buffer) is (3.0 +/- 0.3) = 10(10) M-1 - S-1. As the pH is increased above pH 8.0 the rate constant steadily decreases. The dependence of the rate constant on pH can be explained if ferrocytochrome c has a pK of around 9.2. 3. At 21 degrees C and pH 7.4, the rate constant for the decay of the intermediate is (1.40 +/- 0.15) - 10(5) S-1. This reaction shows no pH dependence in the range 6-2-11.0. 4. A mechanism is proposed whereby the central metal atom of the ferrocytochrome c is oxidased and a thioether bond is reduced. The resulting ferricytochrome c species then slowly develops an absorbance at 606 nm due to the attack of the sulfhydryl group on the haem.     

The stability of ferricytochrome c. Temperature dependence of its NMR spectrum

The temperature dependence of the nuclear magnetic resonance spectrum of horse ferricytochrome c is described. The protein maintains an ordered structure over the temperature range 20 degrees C to 77 degrees C. The temperature dependence of the spectrum of ferricytochrome c arises from a number of causes including the paramagnetism of the ferric ion and protein structural changes. Preliminary analysis of the data show that the region of the protein about Ile-57 is flexible. Comparison of the data with the analogous data for horse ferrocytochrome c reveals that there is a small difference in structure between cytochrome c in its two oxidation states in the region about Ile-57.     

Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome.

Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed.     

Determination of the pK for the acid-induced denaturation of ferrocytochrome c

Optical absorption spectroscopy was used to characterize the acid-induced conformational transition of horse heart ferrocytochrome c in the presence of urea. By using linear extrapolation to zero denaturant concentration, an apparent pK value for denaturation was found to be 0.86 +/- 0.07 at 25 degrees C. Visible absorption spectra in the presence of high urea concentration indicate that the dominant population is a high-spin, five-coordinate form under acidic conditions. Ferricytochrome c, used as a model reference system, shows a linear dependence of pK values versus urea concentration in the range from 0 to 4.1 M. Our data also indicate that even at a pH below 2 the iron-sulfur bond in ferrocytochrome c is present.     

The reaction between the superoxide anion radical and cytochrome c.

1. The superoxide anion radical (O2-) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2- and ferrocytochrome c. 2. At 20 degrees C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4-10(6) M-1. S -1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2- and the form of cytochrome c which exists above pH approximately 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2- reacts with the form present below pH 7.45 with k = 1.4-10(6) M-1 - S-1, the form above pH 7.45 with k = 3.0- 10(5) M-1 - S-1, and the form present above pH 9.2 with k = 0. 3. The reaction has an activation energy of 20 kJ mol-1 and an enthalpy of activation at 25 degrees C of 18 kJ mol-1 both above and below pH 7.45. It is suggested that O2- may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2-6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5-10(5)-5-10(6) M-1 - S-1.     

NMR comparison of prokaryotic and eukaryotic cytochromes c

1H NMR spectroscopy has been used to examine ferrocytochrome c-551 from Pseudomonas aeruginosa (ATCC 19429) over the pH range 3.5-10.6 and the temperature range 4-60 degrees C. Resonance assignments are proposed for main-chain and side-chain protons. Comparison of results for cytochrome c-551 to recently assigned spectra for horse cytochrome c (Wand et al. (1989) Biochemistry 28, 186-194) and mutants of yeast iso-1 cytochrome (Pielak et al. (1988) Eur. J. Biochem. 177, 167-177) reveals some unique resonances with unusual chemical shifts in all cytochromes that may serve as markers for the heme region. Results for cytochrome c-551 indicate that in the smaller prokaryotic cytochrome, all benzoid side chains are rapidly flipping on the NMR time scale. In contrast, in eukaryotic cytochromes there are some rings flipping slowly on the NMR time scale. The ferrocytochrome c-551 undergoes a transition linked to pH with a pK around 7. The pH behavior of assigned resonances provides evidence that the site of protonation is the inner or buried 17-propionic acid heme substituent (IUPAC-IUB porphyrin nomenclature). Conformational heterogeneity has been observed for segments near the inner heme propionate substituent.     

13C-NMR studies of horse ferrocytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of 53 of the 55 methyl resonances of horse ferrocytochrome c have been determined by editing natural abundance 13C spectra according to the number of attached protons, observing the temperature dependence of the chemical shifts, and correlating 13C and proton chemical shifts in two-dimensional spectra. Previous assignments of proton shifts allow 16 of the 13C resonances to be assigned firmly.     

Thermal denaturation of cytochrome c peroxidase: pH dependence

Upon heating cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) at pH 4 and 5, the enzyme precipitates at 41 degrees C and 51 degrees C, respectively. Incubating the enzyme at lower temperatures causes a slow dissociation of the heme from the protein. The heme precipitates, while the apoprotein remains soluble. Between pH 6 and 8, the native enzyme is converted to a low-spin ferric form upon heating. The Soret maximum shifts from 408 to 414 nm. The midpoint of this transition is pH-dependent, with a value of 46 degrees C at pH 6 decreasing to 29 degrees C at pH 8. At high temperatures the 414 nm form is converted to a species which has a 'free heme' spectrum with low absorptivity and Soret maximum at 390 nm. The midpoint temperature of this latter transition is 62 degrees C and 57 degrees C at pH 7 and 8, respectively.     

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.     

Comparison of hydrolysis and esterification behavior of Humicola lanuginosa and Rhizomucor miehei lipases in AOT-stabilized water-in-oil microemulsions: II. Effect of temperature on reaction kinetics and general considerations of stability and productivity

Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RrnL), isolated from commercial preparations of Lipolase and Lipozyme, respectively, were solubilized in AOT-stabilized water-in-oil (w/o) microemulsions in n-heptane and aspects of their hydrolysis and condensation activity examined. The temperature dependence of HIL hydrolysis activity in unbuffered R = 10 microemulsions matched very closely that for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. Apparent activation energies were measured as 13 +/- 2 and 15 +/- 2 kJ mol / respectively. Condensation activity, however, was essentially independent of temperature over the range 5 degrees to 37 degrees C. The stability of HIL over a 30-day period was very good at all pH levels (6.1, 7.2, 9.3) and R values studied (5, 7.5, 10, 20), except when high pHs and low R values were combined. The excellent stability was reflected by the linearity of the productivity profiles which facilitate system optimization. The temperature dependence of RmL hydrolysis activity toward pNPC(4) showed a maximum at 40 degrees C and an apparent E(act) = 20 +/- 2 kJ mol(-1) was calculated based on the linear region of the profile (5 degrees to 40 degrees C). RmL esterification activity showed only a slight dependence on temperature over the studied range (0 degrees to 40 degrees C) and an apparent E(act) = 5 +/- 1 kJ mol(-1) was measured for octyl decanoate synthesis. Both RmL and HIL, therefore, have potential for application in low temperature biotransformations in microemulsion-based media. The stability of RmL over a 30-day period was good in R = 7.5 and R = 10 microemulsions containing pH 6.1 buffer, and this was reflected in the linearity of their respective productivity profiles. RmL stability was markedly poorer at more alkaline pH, however, and proved to be sensitive to relatively small changes in the R value. (c) 1995 John Wiley & Sons, Inc.     

Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR

The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand tau nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine phi nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine tau NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the omega loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center.(ABSTRACT TRUNCATED AT 250 WORDS)     

The salting-out behavior of human plasma fibronectin and its possible correlation with heparin-induced cryoprecipitation of the protein

The solubility of human plasma fibronectin in concentrated ammonium sulfate solutions was measured at pH 7.0 and varying temperatures as well as at 25 degrees C and varying pHs. The salting-out parameters, KS and beta were found to increase linearly with temperature in the range 5 degrees-50 degrees C. KS-pH and beta-pH profiles were found to have maxima at pH 7.0. The dependence of both of the solubility parameters of plasma fibronectin on temperature and pH was thus found to be anomalous. The possibility of a correlation between the heparin-induced cryoprecipitation of fibronectin and the dependence of its solubility parameters on pH and temperature is considered. It is suggested that heparin-induced precipitation of human plasma fibronectin at low temperatures is caused by (i) a cold effect and (ii) conformational change in the protein due to heparin binding.     

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. 3. 1H and 31P nuclear-magnetic-resonance studies on the phosphocarrier protein HPr; tyrosine titration and denaturation studies.

The phosphocarrier protein HPr has been investigated by proton nuclear magnetic resonance (NMR) at 270 MHz in order to evaluate structural properties of the whole molecule and its active site. The titration behaviour of the three tyrosines of the HPr protein was analysed by monitoring the chemical shifts of the aromatic proton resonances of these residues as a function of pH. It was found that the HPr protein contains a lot of slowly exchanging NH backbone protons which suggested a relatively rigid secondary structure of the protein molecule itself although it contains no disulfide bridges. The HPr protein shows a sharp reversible denaturation behaviour at alkaline pH values. Between pH 10.8 and 11.1 two C-2 proton resonance peaks for the single histidine residue could be observed together with abrupt changes in the aromatic and aliphatic absorption region of the HPr protein which are due to chemical exchange processes. The NMR spectrum of the HPr protein is only changed a little upon raising the temperature from 14 degrees C to 70 degrees C. At 76 degrees C all resonances in the spectrum broaden and almost disappear. This process is irreversible.     

Protein stiffening and entropic stabilization in the subdenaturing limit of guanidine hydrochloride

Subdenaturing concentrations of guanidine hydrochloride (GdnHCl) stabilize proteins. For ferrocytochrome c the stabilization is detected at subglobal level with no measured change in global stability. These deductions are made by comparing observed rates of thermally driven ferrocytochrome cHCO reactions with global unfolding rates of ferrocytochrome c measured by stopped flow and NMR hydrogen exchange in the presence of a wide range of GdnHCl concentrations at pH 7, 22 degrees C.     

Reaction of cytochrome c with nitrite and nitric oxide. A model of dissimilatory nitrite reductase.

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.     

13C and proton NMR studies of horse cytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of the 55 methyl groups of horse cytochrome c have been determined over a range of temperatures both in the diamagnetic ferrocytochrome and in the paramagnetic ferricytochrome. Specific assignments of many proton resonances have been published previously and all of the remaining methyl proton resonances are now specifically assigned. The corresponding 13C assignments follow directly, including those of contact shifted 13C resonances which are reported for the first time.     

The temperature dependence of the surface tension of aqueous solutions of plasma proteins.

The surface behavior of aqueous solutions of fibrinogen, transferrin, gamma-globulin and albumin at the liquid-gas interface has been investigated by a modified Wilhelmy technique. The temperature dependence of the surface tension was studied over a temperature range of 20--80 degrees C and a pH range of 2--12. Most pronounced conformational changes of fibrinogen with this technique were found in physiological conditions: 35--45 degrees C and pH 7--8. A conformational change was found for gamma-globulin and transferrin solutions, but at a higher temperature and less pronounced than fibrinogen. Albumin did not undergo conformational transitions to a significant extent.     

The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperature range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be ca. pH 5. At this pH, the specific growth rate was 0.35 h(-1) and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6.     

The downfield resonances in the 1H NMR spectra of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins

Pseudomonas putida and Azotobacter vinelandii ferredoxins each contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. Their polypeptide chains are nearly identical, differing by only 15 residues out of a total of 106. T1 measurements and temperature dependence studies of the 1H NMR spectrum of each ferredoxin demonstrate that all six resolved downfield resonances are near an iron-sulfur center. The five most downfield resonances are shown to arise from protons on cysteinyl beta-carbons by incorporation of cysteine deuterated at the beta-carbon into cell protein. The sixth peak (10.5 ppm) is shown to be a non-cysteinyl proton. This peak resolves into two resonances of approximately equal intensity at temperatures below 15 degrees or above 25 degrees C. A nuclear Overhauser effect observed between the two downfield-most resonances of A. vinelandii ferredoxin indicates that they originate from a geminal pair of beta-cysteinyl protons. An Overhauser effect observed between the resonances at 22.3 and 15.7 ppm, in conjunction with other results, implies that the resonance at 22.3 ppm arises from a beta-proton on the 3Fe-center-bound Cys16, while the resonance at 15.7 ppm arises from Cys45 beta-proton, which is bound to the 4Fe center. The five most downfield resonances are pH-dependent. The sixth peak (10.5 ppm in P. putida ferredoxin) is pH-independent. Possible origins for the observed pH dependencies are discussed.     

Temperature and pH dependence of the proton magnetic hyperfine resonances of cytochrome c peroxidase-cyanide. Evidence for hindered vinyl group rotation as a mediator of the enzymes activity

Proton nmr studies of the hyperfine resonances of cytochrome c peroxidase reveal that two pH-dependent processes can be monitored. One of these is the simple pH titration of a resonance which has been previously assigned to the alpha-vinyl proton at heme position 4. Combined with this proton's temperature dependence, the pH data indicate that the rotational position of vinyl 4 is changing with a pK which is similar to that which regulates the enzyme's activity. The second process, slow on the nmr time scale, occurs above pH 8. This is beyond what is normally considered to be the optimum pH range for cytochrome c peroxidase's activity and we interpret this to indicate a protein conformational change.