Increasing the conformational stability by replacement of heme axial ligand in c-type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.     

Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase.

The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.     

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o3 and its transfer to CuB. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of -1.3+/-0.3 mL mol-1 and enthalpy change of 32+/-1.6 kcal mol-1. Subsequently, a volume increase of 2.9 mL mol-1 with an enthalpy change of -5.3+/-2.5 kcal mol-1 is observed with the lifetime of approximately 250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo3 oxidase indicating that the heme o3/CuB active site dynamics are affected by the redox state of heme b.     

The redox couple of the cytochrome c cyanide complex: the contribution of heme iron ligation to the structural stability, chemical reactivity, and physiological behavior of horse cytochrome c

Contrary to most heme proteins, ferrous cytochrome c does not bind ligands such as cyanide and CO. In order to quantify this observation, the redox potential of the ferric/ferrous cytochrome c-cyanide redox couple was determined for the first time by cyclic voltammetry. Its E0' was -240 mV versus SHE, equivalent to -23.2 kJ/mol. The entropy of reaction for the reduction of the cyanide complex was also determined. From a thermodynamic cycle that included this new value for the cyt c cyanide complex E0', the binding constant of cyanide to the reduced protein was estimated to be 4.7 x 10(-3) L M(-1) or 13.4 kJ/mol (3.2 kcal/mol), which is 48.1 kJ/mol (11.5 kcal/mol) less favorable than the binding of cyanide to ferricytochrome c. For coordination of cyanide to ferrocytochrome c, the entropy change was earlier experimentally evaluated as 92.4 J mol(-1) K(-1) (22.1 e.u.) at 25 K, and the enthalpy change for the same net reaction was calculated to be 41.0 kJ/mol (9.8 kcal/mol). By taking these results into account, it was discovered that the major obstacle to cyanide coordination to ferrocytochrome c is enthalpic, due to the greater compactness of the reduced molecule or, alternatively, to a lower rate of conformational fluctuation caused by solvation, electrostatic, and structural factors. The biophysical consequences of the large difference in the stabilities of the closed crevice structures are discussed.     

Electrostatic stabilization in four-helix bundle proteins.

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.     

Lysines in the amino-terminal alpha-helix are important to the stability of Rhodobacter capsulatus cytochrome c2.

The protein stabilities of wild type and four site-directed mutants of Rhodobacter capsulatus cytochrome c2 have been characterized. The integrity of the cytochrome c2 iron-sulfur environment was ascertained by titration of the 696-nm absorbance band with alkali, and the conformational stability was determined by titration of the 220-nm circular dichroism signal with Gdn-HCl. Analysis of the alkaline transition pK value of K12D (lysine-12 substituted by aspartate) indicated that the K12D iron-sulfur environment was destabilized by 0.6 kcal/mol relative to the wild-type cytochrome c2 at low ionic strength. In contrast, the alkaline transition pK values of K14E (lysine-14 substituted by glutamate), K32E (lysine-32 substituted by glutamate), and K14E/K32E (lysines-14 and -32 substituted by glutamates) were indistinguishable from the wild type, indicating that these substitutions have no effect on the stability of the iron-sulfur environment. Gdn-HCl denaturation of K12D and K14E indicated that both these mutations decreased conformational stability by 1.3 kcal/mol. In contrast, mutant K32E exhibited a small stabilizing effect of 0.2 kcal/mol. Gdn-HCl denaturation of K14E/K32E indicated that this mutation decreased conformational stability by 1.3 kcal/mol, which is consistent with the additive effects of the single charge mutations at positions 14 and 32. The conformational instability of mutants possessing negative charges at position 12 or 14 is best explained by their positioning at the carboxy-terminal region of the amino-terminal alpha-helix of R. capsulatus cytochrome c2. Accordingly, introduction of negatively charged groups into this region appears to destabilize cytochrome c2 through energetically unfavorable interactions with the dipole of the amino-terminal helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Urea-induced sequential unfolding of fibronectin: a fluorescence spectroscopy and circular dichroism study

Fibronectin (FN) is an extracellular matrix (ECM) protein found soluble in corporal fluids or as an insoluble fibrillar component incorporated in the ECM. This phenomenon implicates structural changes that expose FN binding sites and activate the protein to promote intermolecular interactions with other FN. We have investigated, using fluorescence and circular dichroism spectroscopy, the unfolding process of human fibronectin induced by urea in different ionic strength conditions. At any ionic strength, the equilibrium unfolding data are well described by a four-state equilibrium model N <= => I(1) <= =>I(2) <= => U. Fitting this model to experimental values, we have determined the free energy change for the different steps. We found that the N <= => I(1) transition corresponds to a free energy of 10.5 +/- 0.4 kcal/mol. Comparable values of free energy change are generally associated with a partial unfolding of the type III domain. For the I(1) <= => I(2) transition, the free energy change is 7.6 +/- 0.4 kcal/mol at low ionic strength but is twice as low at high ionic strength. This result is consistent with observations indicating that the complete unfolding of the type III domain from partially unfolded forms necessitates about 5 kcal/mol. The third step, I(2) <= => U, which leads to the complete unfolding of fibronectin, corresponds to a free energy change of 14.4 +/- 0.9 kcal/mol at low ionic strength whereas this energy is again twice as low under high ionic strength conditions. This hierarchical unfolding of fibronectin, as well as the stability of the different intermediates controlled by ionic strength demonstrated here, could be important for the understanding of activation of the matrix assembly.     

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Distance dependence and salt sensitivity of pairwise, coulombic interactions in a protein

Histidine pK(a) values were measured in charge-reversal (K78E, K97E, K127E, and K97E/K127E) and charge-neutralization (E10A, E101A, and R35A) mutants of staphylococcal nuclease (SNase) by (1)H-NMR spectroscopy. Energies of interaction between pairs of charges (DeltaG(ij)) were obtained from the shifts in pK(a) values relative to wild-type values. The data describe the distance dependence and salt sensitivity of pairwise coulombic interactions. Calculations with a continuum electrostatics method captured the experimental DeltaG(ij) when static structures were used and when the protein interior was treated empirically with a dielectric constant of 20. The DeltaG(ij) when r(ij) < or = 10 A were exaggerated slightly in the calculations. Coulomb's law with a dielectric constant near 80 and a Debye-Hückel term to account for screening by the ionic strength reproduced the salt sensitivity and distance dependence of DeltaG(ij) as well as the structure-based method. In their interactions with each other, surface charges behave as if immersed in water; the Debye length describes realistically the distance where interactions become negligible at a given ionic strength. On average, charges separated by distances (r(ij)) approximately 5 A interacted with DeltaG(ij) approximately 0.6 kcal/mole in 0.01 M KCl, but DeltaG(ij) decayed to < or =0.10 kcal/mole when r(ij) = 20 A. In 0.10 M KCl, DeltaG(ij) approximately 0.10 kcal/mole when r(ij) = 10 A. In 1.5 M KCl, only short-range interactions with r(ij) < or = 5 A persisted. Although at physiological ionic strengths the interactions between charges separated by more than 10 A are extremely weak, in situations where charge imbalance exists many weak interactions can cumulatively produce substantial effects.     

Calorimetric determination of thermodynamic parameters of reaction reveals different enthalpic compensations of the yeast hexokinase isozymes

The change in enthalpy and rate constants for the reactions of yeast hexokinase isozymes, PI (Hxk1) and PII (Hxk2), was determined at pH 7.6 and 25 degrees C by isothermal titration calorimetry. The reactions were done in five buffer systems with enthalpy of protonation varying from -1.22 kcal/mol (phosphate) to -11.51 kcal/mol (Tris), allowing the determination of the number of protons released during glucose phosphorylation. The reaction is exothermic for both isozymes with a small, but significant (p < 0.0001), difference in the enthalpy of reaction (Delta HR), with an Delta HR of -5.1 +/- 0.2 (mean +/- S.D.) kcal/mol for Hxk1, and an Delta HR of -3.3 +/- 0.3 (mean +/- S.D.) kcal/mol for Hxk2. The Km for ATP determined by ITC was very similar to those reported in the literature for both isozymes. The effect of NaCl and KCl, from 0 to 200 mM, showed that although the rate of reaction decreases with increasing ionic strength, no change in the Delta HR was observed suggesting an entropic nature for the ionic strength. The differences in Delta HR obtained here for both isozymes strongly suggest that, besides glucose phosphorylation, another side reaction such as ATP hydrolysis and/or enzyme phosphorylation is taking place.     

Conformational studies of TMV-RNA: calorimetric and related investigations

Calorimetric measurements of heats arising from transferring Tobacco Mosaic Virus Nucleic Acid (TMV-RNA) from a low to high ionic strength value show that such transfers result in considerable amounts of exothermic heat. Furthermore, the heats measured were found to increase with increasing ionic strength. Velocity sedimentation and viscosity measurements show that in solutions of ionic strength greater than 0.1, the sedimentation coefficient and specific viscosity values of TMV-RNA remain practically unchanged, suggesting that changes in molecular size and hydrodynamic characteristics cannot fully account for the observed heat effects. Optical Rotatory Dispersion (ORD) and Circular Dichroism (CD) measurements of TMV-RNA solutions were made as a function of ionic strength. These studies show that the structure increases with ionic strength. Therefore, conformational changes, mostly as a result of increase in base-pairing and possibly to a smaller extent as a result of changes in base-stacking, appear to be responsible for most, if not all, of the observed heat effects. Energy calculations using the calorimetrically measured heat values show that the energy associated with the conformational changes in TMV-RNA arising from a change in ionic strength from 0.01 to 0.8 is about −2 kcal/mole and this is of the right order of magnitude.     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.     

Perchlorate-induced conformational transition of Staphylococcal nuclease: evidence for an equilibrium unfolding intermediate

The sodium perchlorate-induced conformational transition of Staphylococcal nuclease has been monitored by both circular dichroism (CD) and fluorescence spectroscopy. The perchlorate-induced transition is cooperative as observed by both spectroscopic signals. However, the protein loses only about one-third of its native far-UV CD signal at high perchlorate concentrations, indicating that a significant amount of secondary structure remains in the post-transition state. The remaining CD signal can be further diminished in a cooperative manner by the addition of the strong denaturant, urea. Near-UV CD spectra clearly show that the protein loses its tertiary structure in the perchlorate-induced denatured state. The perchlorate-induced transition curves were fit to the standard two-state model and the standard free energy change and m value of the transition are 2.3kcal/mol and 1.8kcal/(molM), respectively. By comparison, the urea-induced unfolding of Staphylococcal nuclease (in the absence of perchlorate) yields an unfolding free energy change, DeltaG(0,un), of 5.6kcal/mol and an m value of 2.3kcal/(molM). Thus, the thermodynamic state obtained in the post-transition region of perchlorate-induced conformation transition has a significantly lower free energy change, a high content of secondary structure, and diminished tertiary structure. These results suggest that the perchlorate-induced denatured state is a partially folded equilibrium state. Whether this intermediate is relevant to the folding/unfolding path under standard conditions is unknown at this time.     

Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2)

Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.     

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.     

A kinetic analysis of the estrogen receptor transformation.

The rate of the 4 to 5 S estrogen-binding protein (EBP) in vitro transformation was measured by sucrose gradient centrifugation analysis. The temperature-activated 4 to 5 S EBP transformation is found to be highly reproducible without loss of [3H]estradiol-binding activity in a buffer containing an excess of [3H]estradiol, 40 mM Tris, 1 mM dithiothreitol, and 1 M urea at pH 7.4. The presence of [3H]estradiol is necessary for the 4 to 5 EBP transformation. A kinetic analysis of the 4 to 5 EBP transformation shows that it is a bimolecular reaction, the dimerization of the 4 S EBP with a second (similar or dissimilar) monomer or subunit. In buffers containing 0.4 M KCl the apparent second order rate constant is 2.3 plus or minus 0-2 times 10-7 M minus 1 min minus 1 at 28 degrees. The reaction is independent of the initial receptor concentration, suggesting that the 4 S EBP is dissociated into monomeric units in buffers of high ionic strength. In buffers without KCl or with 0.1 M KCl the apparent second order rate constant of receptor transformation increases with decreasing receptor concentration. This suggests that the 4 S EBP is associated weakly with another macromolecule (or macromolecules) in buffers of low ionic strength. The rate of 4 to 5 S EBP transformation shows a 200-fold increase between 0 and 35 degrees. The Arrhenius energy of activation is 21.3 kcal mol minus 1 in buffer without KCl and 19.1 kcal mol minus 1 in buffer with 0.4 M KCl. Following the temperature-activated dimerization, the avidity of binding between the 4 S EBP and its complementary subunit is increased, 0.4 M KCl can no longer cause dissociation, and the 5 S EBP dimer appears. This kinetic analysis indicates that the avidity of binding between the subunits of the estrogen receptor is modulated by estradiol, temperature, and ionic strength. We propose that these interactions of the estrogen receptor's subunits reflect conformational changes involved in receptor activation.