Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria.

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.     

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that     

Reaction of oxidized cytochrome oxidase with cyanide. Effects of pH, cytochrome c and membrane environment

Binding of HCN with ferric beef heart cytochrome oxidase has been studied in submitochondrial particles, as with the enzyme solubilized in detergent or reconstituted into proteoliposomes. Under all conditions, the reaction proceeds via an intermediate and its kinetics can be described by formal parameters Km and kmax in keeping with the Michaelis-type equation. Km of the reaction strongly depends on the enzyme environment; thus it increases 100-1000 fold upon solubilization of cytochrome oxidase but can be subsequently decreased by incorporation of the enzyme in liposomes and by addition of cytochrome c. pH-dependence of the reaction rate shows that, in submitochondrial particles and proteoliposomes as well as in the case of solubilized enzyme supplement with cytochrome c, HCN specifically binds the form of cytochrome oxidase in which a heme-linked ionizable group with pKa 6,5-6,9 is protonated.     

The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH

The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 A). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK(a) values. The results are interpreted in a two-step model for complex formation.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrateferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the K_m (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the ‘association’ rate but also by increasing the ‘dissociation’ rate for bound cytochrome c converting the ‘primary’ (T) site at high salt concentrations into a site similar kinetically to the ‘secondary’ (L) site at low ionic strength. A finite K_m of 170 μM at very high ionic strength indicates a ratio of $\text{K}{}^{\mathrm{\infty }}{}_{\mathrm{<mtext>M</mtext>}}\text{K}{}^0{}_{\mathrm{<mtext>M</mtext>}}$ of about 5000. It is proposed that anions either modify the E¹₀ of cytochrome c bound at the primary (T) site or that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

Protein unfolding in detergents: effect of micelle structure,ionic strength,pH, and temperature

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.     

The dependence of the molecular dynamics of calmodulin upon pH and ionic strength

The mobilities of several fluorescent probes placed at different locations on calmodulin in the absence of Ca2+ have been found to depend upon the charge, ionic strength, and temperature. In general, the time decay of fluorescence anisotropy could be fitted with two rotational correlation times. The shorter of these reflects primarily the motion of the probe itself, while the longer corresponds to the motion of a major portion of the molecule. An increase in ionic strength or a decrease in net charge results in a decrease in the relative amplitude of the shorter correlation time, while an increase in temperature produces an increase in its amplitude. These results are consistent with, and suggest, that an increase in probe mobility accompanies an expansion of the calmodulin molecule under conditions of high electrostatic stress.     

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.     

Porphyrin cytochrome c. pH effects and interaction with cytochrome-c oxidase

1. Porphyrin cytochrome c, the iron-free derivative of cytochrome c, has been used extensively as a fluorescent analog of cytochrome c. It appears as though its fluorescence intensity but not its relative quantum yield is affected by pH in the physiological range; an apparent pK of about 6.2 is found suggesting a histidine close to the porphyrin. 2. The fluorescence intensity of the porphyrin cytochrome c in the presence of cytochrome c oxidase is independent of pH; this suggests that the oxidase has the capacity to control the pK of whichever group is responsible for the pH sensitivity of the free porphyrin cytochrome c. The most likely candidate for this pH-sensitive group is histidine-18. The N-3 nitrogen of this residue forms one of the axial ligands to the iron in the intact cytochrome c but it is uncoordinated in the iron-free derivative.     

The pH dependence of cytochrome a conformation in cytochrome c oxidase.

The pH dependence of the conformation of cytochrome a in bovine cytochrome c oxidase has been studied by second derivative absorption spectroscopy. At neutral pH, the second derivative spectra of the cyanide-inhibited fully reduced and mixed valence enzyme display two Soret electronic transitions, at 443 and 451 nm, associated with cytochrome a. As the pH is lowered these two bands collapse into a single transition at approximately 444 nm. pH titration of the cyanide-inhibited mixed valence enzyme suggests that the transition from the two-band to one-band spectrum obeys the Henderson Hasselbalch relationship for a single protonation event with a transition pKa of 6.6 +/- 0.1. No pH dependence is observed for the spectra of the fully reduced unliganded or CO-inhibited enzyme. Tryptophan fluorescence spectra of the enzyme indicate that no major disruption of protein structure occurs in the pH range 5.5-8.5 used in this study. Resonance Raman spectroscopy indicates that the cytochrome a3 chromophore remains in its ferric, cyanide-bound form in the mixed valence enzyme throughout the pH range used here. These data indicate that the transition observed by second derivative spectroscopy is not due simply to pH-induced protein denaturation or disruption of the cytochrome a3 iron-CN bond. The pH dependence observed here is in good agreement with those observed earlier for the midpoint reduction potential of cytochrome a and for the conformational transition associated with energy transduction in the proton pumping model of Malmstr?m (Malmstr?m, B. G. (1990) Arch. Biochem. Biophys. 280, 233-241). These results are discussed in terms of a model for allosteric communication between cytochrome a and the binuclear ligand binding center of the enzyme that is mediated by ionization of a single group within the protein.     

Swelling studies on the cornea and sclera: the effects of pH and ionic strength.

The biophysical properties of the cornea and sclera depend on the precise maintenance of tissue hydration. We have studied the swelling of the tissues as a function of pH and ionic strength of the bathing medium, using an equilibration technique that prevents the loss of proteoglycans during swelling. Synchrotron x-ray diffraction was used to measure the average intermolecular and interfibrillar spacings, the fibril diameters, and the collagen D-periodicity. We found that both tissues swelled least near pH 4, that higher hydrations were achieved at lower ionic strengths, and that sclera swelled about one-third as much as cornea under most conditions. In the corneal stroma, the interfibrillar spacing increased most with hydration at pH values near 7. Fibril diameters and D-periodicity were independent of tissue hydration and pH at hydrations above 1. Intermolecular spacings in both tissues decreased as the ionic strength was increased, and there was a significant difference between cornea and sclera. Finally, we observed that corneas swollen near pH 7 transmitted significantly more light than those swollen at lower pH levels. The results indicate that the isoelectric points of both tissues are close to pH 4. The effects of ionic strength can be explained in terms of chloride binding within the tissues. The higher light transmission achieved in corneas swollen at neutral pH may be related to the fact that the interfibrillar fluid is more evenly distributed under these conditions.     

Peptoid microsphere coatings: The effects of helicity,temperature, pH,and ionic strength

Peptoids are peptidomimetic oligomers that predominantly harness similarities to peptides for biomimetic functionality. They have potential for use in biomedical applications and biosensors due to resistance to proteolytic degradation and low immunogenicity. The incorporation of chiral, aromatic side chains in the peptoid sequence allows for the formation of distinct secondary structures and self-assembly into supramolecular assemblies, including microspheres. Peptoid microspheres can be coated onto substrates for potential use in biosensor technologies, tissue engineering platforms, and drug-delivery systems. In order to be useful for these applications, the peptoid coatings must be robust under physiological conditions. In this study, we report the effects of various conditions on the peptoid microsphere coatings, including (i) helicity, (ii) temperature, (iii) pH, and (iv) ionic strength. These studies show that microsphere size decreases with increasing peptoid helicity and the positively charged side chains are positioned on the outside of the microspheres. The peptoid microsphere coatings are robust under physiological conditions but degrade in acidic conditions (pH < 7) and at low ionic strengths (<150 μM).     

Physical state of cytochrome oxidase: Relationship between membrane formation and ionic strength

Preparations of lipid-containing and lipid-depleted cytochrome oxidase from beef heart mitochondria through use of Triton non-ionic detergents is described. The lipid-containing oxidase is shown to form either a membranous or dispersed state. The dispersed state requires the presence of salt whereas the membranous condition is obtained by reducing the ionic strength. The membranous form shows low activity in comparison to the dispersed form. Typical unit membrane structure can be shown in sections of the membranous oxidase or in reaggregated membranous oxidase even though the lipid content is as low as eight percent. Both the dispersed and membranous oxidase show 50-Å globules by negative staining whereas the lipid-free oxidase shows strings of 20 Å diameter.     

Cartilage electromechanics--I. Electrokinetic transduction and the effects of electrolyte pH and ionic strength

Articular cartilage contains a high fixed charge density under physiological conditions associated primarily with the ionized proteoglycan molecules of the extracellular matrix. Oscillatory compression of cartilage using physiological loads produces electrical potentials that have been shown previously to be the result of an electrokinetic (streaming) transduction mechanism. We have now observed two additional electromechanical phenomena not previously seen in cartilage or other soft tissues: 'streaming current' and 'current-generated stress'. Sinusoidal mechanical compression induced a sinusoidal streaming current density through cartilage disks when the Ag/AgCl electrodes that were used to compress the cartilage were shorted together externally. Conversely, a sinusoidal current density applied to the tissue generated a sinusoidal mechanical stress within the tissue. Both these phenomena were found to be consistent with the same electrokinetic transduction mechanism responsible for the streaming potential. Changes in the measured streaming potential response that resulted from modification of bath ionic strength and pH have provided additional insights into the molecular origins of these transduction processes. Finally, we have now observed streaming potentials in living cartilage maintained in organ culture, as well as in previously frozen tissue.