Sedimentation equilibrium studies on the interaction between cytochrome c and cytochrome c peroxidase

The interaction between cytochrome c and cytochrome c peroxidase was investigated using sedimentation equilibrium at pH 6,20 degrees C, in a number of buffer systems varying in ionic strength between 1 and 100 mM. Between 10 and 100 mM ionic strengths, the sedimentation of the individual proteins was essentially ideal, and sedimentation equilibrium experiments on mixtures of the two proteins were analyzed assuming ideal solution behavior. Analysis of the distribution of mixtures of cytochrome c and cytochrome c peroxidase in the ultracentrifuge cell based on a model involving the formation of a 1:1 cytochrome c-cytochrome c peroxidase complex gave values of the equilibrium dissociation constant ranging from 2.3 +/- 2.7 microM at 10 mM ionic strength to infinity (no detectable interaction) at 100 mM ionic strength. Attempts to determine the presence of complexes involving two cytochrome c molecules bound to cytochrome c peroxidase were inconclusive.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.

The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.     

Multidimensional diffusion modes and collision frequencies of cytochrome c with its redox partners

We have determined the modes and rates of cytochrome c diffusion as well as the collision frequencies of cytochrome c with its redox partners at the surface of the isolated, mitochondrial inner membrane over a broad range (0-150 mM) of ionic strengths. Using fluorescence recovery after photobleaching, resonance energy transfer, and direct binding assay, we determined that the diffusion coefficient of cytochrome c is independent of its concentration and quantity bound to the inner membrane, that the distance of cytochrome c from the membrane surface increases with increasing ionic strength, and that there is no significant immobile fraction of cytochrome c on the membrane regardless of ionic strength. The rate of cytochrome c diffusion increases while its mode of diffusion changes progressively from lateral to three-dimensional with increasing ionic strength. At physiological ionic strength (100-150 mM), the diffusion of cytochrome c is three-dimensional with respect to the surface of the inner membrane with a coefficient of 1.0 x 10(-6) cm2/s, and little, if any cytochrome c is bound to the membrane regardless of its concentration. Furthermore, as ionic strength is raised from zero to 150 mM, the cytochrome ckd for the inner membrane increases, its mean occupancy time on the inner membrane to collide with a redox partner (tau) decreases, and its diffusion-based collision frequencies with its redox partners decrease. These data reveal the significance of both diffusion and concentration (affinity) of cytochrome c near the surface of the inner membrane in the control of the collision frequency of cytochrome c with its redox partners.     

Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes.

Membrane association of cytochrome c (cyt c) was monitored by the efficiency of resonance energy transfer from a pyrene-fatty acid containing phospholipid derivative (1-palmitoyl-2[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine (PPHPC)) to the heme of cyt c. Liposomes consisted of 85 mol% egg phosphatidylcholine (egg PC), 10 mol% cardiolipin, and 5 mol% PPHPC. Cardiolipin was necessary for the membrane binding of cyt c over the pH range studied, from 4 to 7. In accordance with the electrostatic nature of the membrane association of cyt c at neutral pH both 2 mM MgCl2 and 80 mM NaCl dissociated cyt c from the vesicles completely. At neutral pH also adenine nucleotides in millimolar concentrations were able to displace cyt c from liposomes, their efficiency decreasing in the sequence ATP > ADP > AMP. In addition, both CTP and GTP were equally effective as ATP. The detachment of cyt c from liposomes by nucleotides is likely to result from a competition between cardiolipin and the nucleotides for a common binding site in cyt c. When pH was decreased to 4 there was a small yet significant increase in the apparent affinity of cyt c to cardiolipin containing liposomes. Notably, at pH 4 the above nucleotides as well as NaCl and MgCl2 were no longer able to dissociate cyt c and, on the contrary, they slightly enhanced the quenching of pyrene fluorescence by cyt c. The above results do suggest that the membrane association of cyt c at acidic pH was non-ionic and presumably due to hydrogen bonding. The pH-dependent binding of cyt c to membranes was fully reversible. Accordingly, in the presence of sufficient concentrations of either nucleotides or salts rapid detachment and membrane association of cyt c could be induced by varying pH between neutral and acidic values, respectively.     

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.     

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

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.     

Cytochrome C interaction with cardiolipin/phosphatidylcholine model membranes: effect of cardiolipin protonation

Resonance energy transfer between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cytochrome c (cyt c) as an acceptor has been employed to explore the protein binding to model membranes, composed of phosphatidylcholine and cardiolipin (CL). The existence of two types of protein-lipid complexes has been hypothesized where either deprotonated or partially protonated CL molecules are responsible for cyt c attachment to bilayer surface. To quantitatively describe cyt c membrane binding, the adsorption model based on scaled particle and double layer theories has been employed, with potential-dependent association constants being treated as a function of acidic phospholipid mole fraction, degree of CL protonation, ionic strength, and surface coverage. Multiple arrays of resonance energy transfer data obtained under conditions of varying pH, ionic strength, CL content, and protein/lipid molar ratio have been analyzed in terms of the model of energy transfer in two-dimensional systems combined with the adsorption model allowing for area exclusion and electrostatic effects. The set of recovered model parameters included effective protein charge, intrinsic association constants, and heme distance from the bilayer midplane for both types of protein-lipid complexes. Upon increasing CL mole fraction from 10 to 20 mol % (the value close to that characteristic of the inner mitochondrial membrane), the binding equilibrium dramatically shifted toward cyt c association with partially protonated CL species. The estimates of heme distance from bilayer center suggest shallow bilayer location of cyt c at physiological pH, whereas at pH below 6.0, the protein tends to insert into membrane core.     

Characterization of the interaction of cytochrome c and mitochondrial ubiquinol-cytochrome c reductase

Characterization of the steady state kinetics of reduction of horse ferricytochrome c by purified beef ubiquinol-cytochrome c reductase, employing 2,3-dimethoxy-5-methyl-6-decylbenzoquinol as reductant, has shown that: 1) the dependence of the reaction on quinol and on ferricytochrome c concentration is consistent with a ping-pong mechanism; 2) the pH optimum of the reaction is near 8.0; 3) the effect of ionic strength on the apparent Km and the TNmax of the reaction for the native cytochrome c is small, and at higher cytochrome c concentrations substrate inhibition is observed; 4) the effect of ionic strength on the kinetic parameters for the reaction of 4-carboxy-2,6-dinitrophenyllysine 27 horse cytochrome c is much larger than for the native protein; and 5) competitive product inhibition is also observed with a Ki consistent with the binding affinity of ferrocytochrome c for Complex III, as determined by gel filtration. In addition, direct binding measurements demonstrated that ferricytochrome c binds more tightly than the reduced protein to Complex III under low ionic strength conditions and that under these conditions more than one molecule of cytochrome c is bound per molecule of Complex III. Exchange of Complex III into a nonionic detergent decreases this excess nonspecific binding. Measurement of the rates of dissociation of the oxidized and reduced 1:1 complexes of cytochrome c and Complex III by stopped flow was consistent with the disparity of binding affinities, the dissociation rate constant for ferrocytochrome c being about 5-fold higher than that for the ferric protein. A model which accounts for the properties of this system is described, assuming that cytochrome c bound to noncatalytic sites on the respiratory complex decreases the catalytic site binding constant for the substrate.     

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.     

Ionic strength effects on cytochrome aa3 kinetics

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa3 can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa3 increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa3 complex limits the ferrocytochrome c association to the low affinity site. 2. At low ionic strength, the reduction of cytochrome c-aa3 complex by ferrocytochrome c1 proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c1 and aa3. 3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa3 complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c. 4. Dissociation rates of both high and low affinity sites on cytochrome aa3 were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 microM for the high and low affinity site, respectively.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

Isolation of a multiprotein complex containing cytochrome b and c1 from Neurospora crassa mitochondria by affinity chromatography on immobilized cytochrome c. Difference in the binding between ferricytochrome c and ferrocytochrome c to the multiprotein complex.

A multiprotein complex which contains in equimolar amounts two cytochromes b (Mr each about 27,000), one cytochrome c1 (Mr 31,000) and six subunits without known prosthetic groups (Mr 8000, 12,000, 14,000, 45,000, 45,000, and 50,000) has been isolated from the mitochondrial membranes of Neurospora crassa by affinity chromatography on immobilized cytochrome c. The chromatographic separation was based upon the specific binding of the complex to ferricytochrome c coupled to Sepharose and its specific release upon conversion of the coupled ferricytochrome c into ferrocytochrome c using ascorbate as a reductant. The chromatography was performed in the presence of the nonionic detergent Triton X-100 at low ionic strengths. A monodisperse preparation of the multiprotein complex was obtained which was used for binding studies with cytochrome c from Neurospora crassa, horse heart and Saccaromyces cerevisiae. At low ionic strength (20 mM Trisacetate) and slightly alkaline pH (pH 7 to 8), more than one molecule of ferricytochrome c were bound to the isolated multiprotein complex with dissociation constants below 1 x 10(-7) M. One of these bindings appeared different from the others, since its high affinity was preserved at an ionic strength at which the affinities of the other bindings decreased. Furthermore, the affinity of only this binding decreased upon reduction of cytochrome c. It is suggested that this binding is at or near the functionally active site(s) of the mulipprotein complex.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Unbinding of oxidized cytochrome c from photosynthetic reaction center of Rhodobacter sphaeroides is the bottleneck of fast turnover

To understand the details of rate limitation of turnover of the photosynthetic reaction center, photooxidation of horse heart cytochrome c by reaction center from Rhodobacter spheroides in detergent dispersion has been examined by intense continuous illumination under a wide variety of conditions of cytochrome concentration, ionic strength, viscosity, temperature, light intensity, and pH. The observed steady-state turnover rate of the cytochrome was not light intensity limited. In accordance with recent findings [Larson, J. W., Wells, T. A., and Wraight, C. A. (1998) Biophys. J. 74 (2), A76], the turnover rate increased with increasing bulk ionic strength in the range of 0-40 mM NaCl from 1000 up to 2300 s(-)(1) and then decreased at high ionic strength under conditions of excess cytochrome and ubiquinone and a photochemical rate constant of 4500 s(-)(1). Furthermore, we found the following: (i) The contribution of donor (cytochrome c) and acceptor (ubiquinone) sides as well as the binding of reduced and the release of oxidized cytochrome c could be separated in the observed kinetics. At neutral and acidic pH (when the proton transfer is not rate limiting) and at low or moderate ionic strength, the turnover rate of the reaction center was limited primarily by the low release rate of the photooxidized cytochrome c (product inhibition). At high ionic strength, however, the binding rate of the reduced cytochrome c decreased dramatically and became the bottleneck. The observed activation energy of the steady-state turnover rate reflected the changes in limiting mechanisms: 1.5 kcal/mol at 4 mM and 5.7 kcal/mol at 100 mM ionic strength. A similar distinction was observed in the viscosity dependence of the turnover rate: the decrease was steep (eta(-)(1)) at 40 and 100 mM ionic strengths and moderate (eta(-)(0.2)) under low-salt (4 mM) conditions. (ii) The rate of quinone exchange at the acceptor side with excess ubiquinone-30 or ubiquinone-50 was higher than the cytochrome exchange at the donor side and did not limit the observed rate of cytochrome turnover. (iii) Multivalent cations exerted effects not only through ionic strength (screening) but also by direct interaction with surface charge groups (ion-pair production). Heavy metal ion Cd(2+) bound to the RC with apparent dissociation constant of 14 microM. (iv) A two-state model of collisional interaction between reaction center and cytochrome c together with simple electrostatic considerations in the calculation of rate constants was generally sufficient to describe the kinetics of photooxidation of dimer and cytochrome c. (v) The pH dependence of cytochrome turnover rate indicated that the steady-state turnover rate of the cytochrome under high light conditions was not determined by the isoelectric point of the reaction center (pI = 6. 1) but by the carboxyl residues near the docking site.     

Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy

The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.     

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans

We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al. [(1999) J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans. Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca(2+), or higher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formation with redox partners were performed in the presence of Ca(2+) in order to simplify the equilibria that had to be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K(d) values of 0.8 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface. For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range. We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase.