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PubMed1999

Theoretical and experimental investigations of electrostatic effects on acetylcholinesterase catalysis and inhibition.

The role of electrostatics in the function of acetylcholinesterase (AChE) has been investigated by both theoretical and experimental approaches. Second-order rate constants (kE = k(cat)/Km) for acetylthiocholine (ATCh) turnover have been measured as a function of ionic strength of the reaction medium for wild-type and mutant AChEs. Also, binding and dissociation rate constants have been measured as a function of ionic strength for the respective charged and neutral transition state analog inhibitors m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenone (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that kE for ATCh turnover is rate limited by terminal binding events. Comparison of binding rate constants for TMTFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of wild-type and mutant AChEs have been calculated via a model that utilizes the structure of T. californica AChE, a spherical ligand, and energy terms that account for electrostatic and van der Waals interactions and chemical potential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barriers for ligand release give linear free-energy correlations with log(kE) for substrate turnover, which supports the general correctness of the computational model.

Acetophenones↗
PubMed1990

A precise analytical method for calculating the electrostatic energy of macromolecules in aqueous solution.

A new method for calculating the total electrostatic free energy of a macromolecule in solution is presented. It is applicable to molecules of arbitrary shape and size, including membranes or macromolecular assemblies with substrate molecules and ions. The method is derived from integrating the energy density of the electrostatic field and is termed the field energy method. It is based on the dielectric model, in which the solute and the surrounding water are regarded as different continuous dielectrics. The field energy method yields both the interaction energy between all charge pairs and the self energy of single charges, effectively accounting for the interaction with water. First, the dielectric boundary and mirror charges are determined for all charges of the solute. The energy is then given as a simple function of the interatomic distances, and the standard atomic partial charges and volumes. The interaction and self energy are shown to result from three-body and pairwise interactions. Both energy terms explicitly involve apolar atoms, revealing that apolar groups are also subject to electrostatic forces. We applied the field energy method to a spherical model protein. Comparison with the Kirkwood solution shows that errors are within a small percentage. As a further test, the field energy method was used to calculate the electrostatic potential of the protein superoxide dismutase. We obtained good agreement with the result from a program that implements the numerical finite difference algorithm. The field energy method provides a basis for energy minimization and dynamics programs that account for the solvent and screening effect of water at little computational expense.

Electricity↗
PubMed1997

Significant role of electrostatic interactions for stabilization of protein assemblies.

Contribution of electrostatic interactions to stability of BPTI orthorhombic, pig-insulin cubic crystals, and horse L ferritin crystals was evaluated with numerical calculation of Poisson-Boltzmann equation based on a dielectric model. The stability of a ferritin molecule (24-mer) composed of 24 subunits was also evaluated. It was found that the surface charge-charge interactions at separation distances (< 5 A) were insensitive to variations in the ionic strength, and thus stabilized assembled states of the proteins (i.e., crystalline state and oligomeric state). It was also revealed that the charge density and the packing of the protein crystals were largely responsible for the ionic strength dependence of the crystal stability. The stability of the 5PTI crystalline state with a high charge density drastically increased as the concentration of the solvent ions increased. In contrast, that of the insulin crystal with a low charge density and large solvent region was insensitive to changes in the ionic concentration. The electrostatic interaction between ferritin 24-mers was attributed to two salt bridges mediated by Cd ion. For the stability of the ferritin 24-mer, which is evolutionally designed, the electrostatic stabilization between the subunits was attributed to polar bonds such as buried salt bridges or hydrogen bonds, which occasionally yielded more than 5 kcal/mol and were numerous and very strong compared with the bonds between molecules in the 5PTI and 9INS crystals. By analyzing the atomic charge-charge interactions in detail, it was found that charge pairs separated by less than 3 A, such as hydrogen bonds, dominantly stabilize the assembled states, and that pairs 3 to 5 A apart were also important. The stability of the assembled states evaluated by the total EET was determined by the fine balance between the two competing contributions arising from the stabilizing atoms and the destabilizing atoms. Changes of the ASA and hydration free energy were also evaluated in accordance with the process of the subunit assembly. The change of hydration free energy, which was very large (i.e., approximately +100 kcal/mol/subunit) and unfavorable for the assembly, was proportional to the electrostatic hydration energy (i.e., Born energy change in the hydration process). Hydrophobic groups were likely to appear more frequently than hydrophilic groups at the interfaces. This study offers a method which can improve the stability of protein crystals by introducing polar or charged residues that are properly designed to form specific hydrogen bonds or salt bridges between neighboring protein molecules. This method is also applicable to crystallography, because it improves refinement of protein structures in crystals by taking the inter-protein interactions into account.

Algorithms↗
PubMed1997

Modeling of anti-nucleosome immunoglobulin Fv domains: analysis of electrostatic interactions.

Three-dimensional structural models of six murine anti-(H2A-H2B) monoclonal autoantibody variable fragments were built by comparative molecular modeling using the COMPOSER software. Analysis of the antibody combining sites is based on the hypothesis that ionic and/or electrostatic interactions predominate in antigen antibody binding, as suggested by the cationic nature of histones and the amino acid sequences of the antibody hypervariable regions. The study of the electrostatic potentials of their combining site surfaces, computed with the MOLCAD software, and the comparison with the electrostatic potentials of 13 selected control mAbs show the lack of a unique electrostatic pattern. One group of three mAbs expresses a strong and large electronegative area, supporting the hypothesis that ionic interactions predominate in antigen recognition. The second group, containing the other three mAbs, exhibits an alternation of electropositive and electronegative areas. All, however, present a localized electronegative area in the vicinity of H-CDR1 and H-CDR2 loops that is generated by the presence of at least one acidic residue. The model suggesting that the binding activity may depend on charged residues at the same site is reminiscent of what was previously reported in anti-DNA mAbs. In addition, the alternation of electropositive areas and electronegative areas in second group mAbs is also frequently observed in certain anti-DNA mAbs. These data argue for the existence of relationships between these two autoantibody populations and suggest that they share a common immunogenic particle formed by anionic and cationic components, such as a nucleosome.

Amino Acid Sequence↗
PubMed2000

Phase detection of electrostatic force by AFM with a conductive tip

Electrostatic force measurements were performed by the Kelvin probe force microscopy, which consists of the atomic force microscopy and a conductive tip. Sample surface potential was evaluated through the electrostatic force, which works between the sample and the tip when we apply an AC bias at a frequency f(s) with a DC offset. If the DC offset voltage is equal to the surface potential difference between the sample and the tip, the amplitude of the f(s) component in the electrostatic force becomes zero, and the phase of the f(s) component jumps 180 degrees there. We found that the complementary use of the amplitude and phase signals improved the accuracy of the determination of the surface potential. We measured both the work functions of some metals and the surface Fermi levels of an InAs layers on (1 0 0) and (1 1 0) GaAs substrates based on the phase detection method of the electrostatic force.

Journal Article↗
PubMed2000

Electrostatic interaction between stereocilia: II. Influence on the mechanical properties of the hair bundle.

This paper is based on our model [Dolgobrodov et al., 2000. Hear. Res., submitted for publication] in which we examine the significance of the polyanionic surface layers of stereocilia for electrostatic interaction between them. We analyse how electrostatic forces modify the mechanical properties of the sensory hair bundle. Different charge distribution profiles within the glycocalyx are considered. When modelling a typical experiment on bundle stiffness measurements, applying an external force to the tallest row of stereocilia shows that the asymptotic stiffness of the hair bundle for negative displacements is always larger than the asymptotic stiffness for positive displacements. This increase in stiffness is monotonic for even charge distribution and shows local minima when the negative charge is concentrated in a thinner layer within the cell coat. The minima can also originate from the co-operative effect of electrostatic repulsion and inter-ciliary links with non-linear mechanical properties. Existing experimental observations are compared with the predictions of the model. We conclude that the forces of electrostatic interaction between stereocilia may influence the mechanical properties of the hair bundle and, being strongly non-linear, contribute to the non-linear phenomena, which have been recorded from the auditory periphery.

Animals↗
PubMed2001

Electrostatics calculations: recent methodological advances and applications to membranes.

Calculations of electrostatic energies and forces are at the heart of the theoretical modeling of biological molecules. During the past year, new methods for accurately treating electrostatic interactions have been developed for all-atom simulations and for modeling systems in which the uninteresting part of the system, for example, the solvent, is represented implicitly. Recent applications of electrostatic energy calculations have revealed new principles concerning the role of electrostatics in peptide binding to membranes and ion transport across membranes through protein channels.

Artifacts↗
PubMed2003

Electrostatic energy analysis of 8-oxoguanine DNA lesion-molecular dynamics study.

One nanosecond molecular dynamics (MD) simulation was performed for two DNA segments each composed of 30 base pairs. In one DNA segment the native guanines at nucleotides positions 17 and 19 were replaced with two 8-oxoguanines (8-oxoG) (8-oxoG is mutagenic DNA oxo-lesion). The analysis of results was focused on the electrostatic energy that is supposed to be significant factor causing the disruption of DNA base stacking in DNA duplex and may also serve as a signal toward the repair enzyme informing the presence of the lesion. The repulsive interaction between 8-oxoG and the entire DNA molecule was observed, which caused the extrahelical position of 8-oxoG (position 19). The repulsive electrostatic interaction between both 8-oxoG lesions contributed to the flipping out of one 8-oxoG and to the local instability of the lesioned DNA region. The electrostatic potential at the surface of DNA close to the lesions has more negative value than the same region on the native DNA. This electrostatic potential may signal presence of the lesion to the repair enzyme. In the simulation of native DNA segment, no significant structural changes were observed and B-DNA structure was well preserved throughout the MD simulation.

DNA↗
PubMed2001

Chromatographic probing of electrostatic potential.

Electrostatic potential in the vicinity of the surface of a cation-exchange resin has been evaluated by modeling chromatographic retention. Binary mixtures of K+ and its crown ether complex in methanol are used as mobile phases, and two types of solutes, that is, cationic and crown ether probes, have been examined. The cationic probes show the sigmoidal retention changes with increasing concentration of a crown ether incorporated into the mobile phase, whereas crown ether probes give retention maximums. The model derived from the Poisson-Boltzmann theory well explains these specific changes in probe retention and gives the electrostatic potential at the closest approach of each probe molecule. The closest approaches for probe molecules correlate well with their molecular sizes. In addition, changes in retention of cationic probes also correlate well with the electrostatic potential changes at the closest approaches of probe molecules, indicating that simple sensing of the electrostatic potential is feasible using probe retention. The reduction of crown ether complexation occurs in the vicinity of the cation-exchange resin surface and causes the specific retention behaviors of crown ether probes in the mobile-phase systems composed of K+ and its complex with a modifier crown ether.

Journal Article↗
PubMed2004

Electrostatic contributions to protein retention in ion-exchange chromatography. 1. Cytochrome C variants.

Among the factors that modulate protein interactions, several protein structural properties, such as size, shape, and charge distribution, may play significant roles. In this work, we investigate the influence of protein structure on binding in ion-exchange chromatography, in which electrostatic interactions are dominant. Chromatographic experiments show separation of cytochrome c variants with a limited number of sequence differences to be feasible. To probe the molecular basis for this behavior, protein-adsorbent electrostatic interactions were modeled in the context of continuum electrostatics accounting for the full 3D protein structure. Protein retention was modeled by averaging over all protein-adsorbent configurations using the full accessible surface of the protein. The electrostatic interaction free energy distribution shows that configurations in which numerous positive protein charges are close to the cation exchanger functional groups produce the most favorable binding. The calculated binding equilibrium constant, found by averaging over the full 3D configurational space, captures the chromatographic differentiation of closely related cytochrome c variants. To obviate the need for full sampling of protein configurations, calculations of interaction free energies at short protein-adsorbent separation distances or of protein surface potentials were found to yield reasonable semiquantitative descriptions of the retention trends.

Adsorption↗
PubMed1993

Electron-transfer kinetics and electrostatic properties of the Rhodobacter sphaeroides reaction center and soluble c-cytochromes.

The kinetics of electron transfer between the Rhodobacter sphaeroides R-26 reaction center and nine soluble c-cytochromes have been analyzed and compared to the patterns of the surface electrostatic potentials for each of the proteins. Characteristic first-order electron-transfer rates for 1:1 complexes formed at low ionic strength between the reaction center and the different c-cytochromes were identified and found to vary by a factor of almost 100, while second-order rates were found to differ by greater than 10(6). A correlation was found between the location of likely electrostatic interaction domains on each cytochrome and its characteristic rate of electron transfer. The interaction domains were identified by mapping electrostatic potentials, calculated from the Poisson-Boltzmann equation, onto simulated "encounter surfaces" for each of the cytochromes and the reaction center. For the reaction center, the c-cytochrome binding domain was found to have almost exclusively net negative potential (< -3 kT) and to be shifted slightly toward the M-subunit side of the reaction center. The location of interaction domains of complementary, positive potential (> 3 kT) differed for each cytochrome. The correspondence between electrostatic, structural, and kinetic properties of 1:1 reaction center-cytochrome complexes leads to a proposed mechanism for formation of reaction center-cytochrome electron-transfer complexes that is primarily driven by the juxtaposition of regions of delocalized complementary potential. In this mechanism the clustering of charged residues is of primary importance and not the location of specific residues. A consequence of this mechanism is that many different sets of charge distributions are predicted to be capable of stabilizing a specific configuration for a reaction center-cytochrome complex. This mechanism for reaction center association with water-soluble c-cytochromes fits molecular recognition mechanisms proposed for c-cytochromes in nonphotosynthetic systems. In general, the kinetic scheme for reaction center driven cytochrome oxidation was found to vary between a simple two-state model, involving cytochrome in free and reaction center bound states, and a three-state model, that includes cytochrome binding in kinetically competent ("proximal") and incompetent ("distal") modes. The kinetically incompetent mode of cytochrome binding is suggested not to be an intrinsic feature of the reaction center-cytochrome association but is likely to be due to variation in the physical state of the reaction center.

Animals↗
PubMed1994

Electrostatic potential surface analysis of the transition state for AMP nucleosidase and for formycin 5'-phosphate, a transition-state inhibitor.

AMP nucleosidase hydrolyzes the N-glycosidic bond of AMP to yield adenine and ribose 5-phosphate. Kinetic isotope effects have been used to establish an experimentally based transition-state structure for the native enzyme and a Vmax mutant [Mentch, F., Parkin, D. W., & Schramm, V. L. (1987) Biochemistry 26, 921-930; Parkin, D. W., Mentch, F., Banks, G. A., Horenstein, B. A., & Schramm, V. L. (1991) Biochemistry 30, 4586-4594]. The transition states are characterized by weak reaction coordinate bonds to C1' and substantial carbocation character in the ribose ring. The N9-C1' bond to the leaving group is nearly broken and the adenine ring is protonated at the transition state. Formycin 5'-phosphate and other purine nucleoside 5'-phosphate analogues with syn-glycosyl torsion angles bind better than substrate, supporting a syn configuration in the enzyme-substrate complex and presumably in the transition state [Giranda, V. L., Berman, H. M., & Schramm, V. L. (1988) Biochemistry 27, 5813-5818]. Access to a geometric model of the transition state permits the analysis of its molecular electrostatic potential surface as enforced by the enzyme. Comparison of the molecular electrostatic potential surfaces for AMP, formycin 5'-phosphate, and the transition state reveals a striking similarity in the surface charges of formycin 5'-phosphate and the transition state. The enzyme-stabilized transition state for AMP hydrolysis is characterized by new positive electrostatic potential in the adenine ring as a result of protonation by the enzyme. This is closely matched by the protonated pyrazole ring of formycin 5'-phosphate. The molecular electrostatic potential surfaces of formycin 5'-phosphate and the transition state for AMP are similar and are likely to be a factor in the Km/Ki value of > 10(3) for formycin 5'-phosphate as a transition-state inhibitor of AMP nucleosidase.

Electricity↗
PubMed2001

Role of charged residues in the catalytic mechanism of hepatitis C virus NS3 protease: electrostatic precollision guidance and transition-state stabilization.

Maturational cleavage of the hepatitis C virus polyprotein involves the viral chymotrypsin-like serine protease NS3. The substrate binding site of this enzyme is unusually flat and featureless. We here show that NS3 has a highly asymmetric charge distribution that is characterized by strong positive potentials in the vicinity of its active site and in the S5/S6 region. Using electrostatic potential calculations, we identified determinants of this positive potential, and the role of six different residues was explored by site-directed mutagenesis. Mutation of residues in the vicinity of the active site led to changes in k(cat) values of a peptide substrate indicating that basic amino acids play a role in the stabilization of the transition state. Charge neutralization in the S5/S6 region increased the K(m) values of peptide substrates in a manner that depended on the presence of negatively charged residues in the P5 and P6 positions. K(i) values of hexapeptide acids spanning P6-P1 (product inhibitors) were affected by charge neutralization in both the active site region and the S5/S6 region. Pre-steady-state kinetic data showed that the electrostatic surface potential is used by this enzyme to enhance collision rates between peptidic ligands and the active site. Calculations of the interaction energies of protease-substrate or protease-inhibitor complexes showed that electrostatic interaction energies oppose the formation of a tightly bound complex due to an unfavorable change in the desolvation energy. We propose that desolvation costs are minimized by avoiding the formation of individual ion pair interactions through the use of clusters of positively charged residues in the generation of local electrostatic potentials.

Amino Acid Sequence↗
PubMed2001

Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: the role of the thrombin anion-binding exosite-I.

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75), Arg(77a), Lys(81), Lys(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (deltaG(degrees)b) by each residue varied from 1.9 kJ mol(-)(1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A., Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (delta deltaG(degrees) int) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (deltadeltaG(degrees)int 0.6 kJ mol(-1)) and h-Glu(58)/Arg(77a) (deltadeltaG(degrees)int 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) (deltadeltaG(degrees)int 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (deltadeltaG(degrees)int 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.

Amino Acid Substitution↗
PubMed1982

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor.

The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.

Animals↗
PubMed1984

Hydrophobic and electrostatic interactions between adrenocorticotropin-(1-24) -tetracosapeptide and lipid vesicles. Amphiphilic primary structures.

Hydrophobic photolabeling with 3-(trifluoromethyl) -3-(m-[125I]iodophenyl) diazirine ( [125I]TID [Brunner, J., & Semenza , G. (1981) Biochemistry 20, 7174-7182] ) and equilibrium dialysis were used to study hydrophobic and electrostatic interactions between three adrenocorticotropin fragments and liposomes prepared from mixtures of phosphatidylcholine with phosphatidic acid or phosphatidylserine. Corticotropin-(1-10)-decapeptide (ACTH1-10, net charge 0) formed hydrophobic clusters with [125I]TID in aqueous solutions at peptide concentrations above 1 microM but did not interact appreciably with neutral or anionic liposomes. Corticotropin -(11-24)-tetradecapeptide ( ACTH11 -24, net charge 6+) reacted electrostatically with anionic liposomes but showed no hydrophobic interactions. Corticotropin-(1-24)-tetracosapeptide (ACTH1-24, net charge 6+), a covalent combination of the two fragments, exhibited both hydrophobic and electrostatic interactions with lipid vesicles. Edman degradation and chymotryptic hydrolysis of labeled ACTH1-24 revealed that the hydrophobic interaction involved the N-terminal decapeptide "message" segment (corresponding to ACTH1-10) which entered the membrane and that the electrostatic interaction was caused by the C-terminal tetradecapeptide "address" segment (corresponding to ACTH11 -24) which remained on the aqueous membrane surface. This surface is in complete analogy to that reported for dynorphin- (1-13)-tridecapeptide by Gysin and Schwyzer [ Gysin , B., & Schwyer , R. (1983) FEBS Lett. 158, 12-16; Gysin , B., & Schwyzer , R. (1983) Arch. Biochem. Biophys. 225, 467-474]: in both cases, the specific, hydrophobic membrane interaction of the "message" critically depended on the presence of the hydrophilic "address". The results reported here were consistent with those obtained by infrared attenuated total reflection spectroscopy [ Gremlich , H.-U., Fringeli , U.-P., & Schwyzer , R. (1983) Biochemistry 22, 4257-4263] and were crucial for their interpretation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1987

Electrostatic effect upon association of reduced nicotinamide adenine dinucleotide and equine liver alcohol dehydrogenase.

The rate of association of equine liver alcohol dehydrogenase and its coenzymes exhibits a large pH dependence with slower rates at basic pH and an observed kinetic pKa value of approximately 9-9.5. This pH dependence has been explained by invoking local active site electrostatic effects which result in repulsion of the negatively charged coenzyme and the ionized hydroxyl anion form of the zinc-bound water molecule. We have examined a simpler hypothesis, namely, that the pH dependence results from the electrostatic interaction of the coenzyme and the enzyme which changes from an attractive interaction of the negatively charged coenzyme and the positively charged enzyme to a repulsive interaction between the two negatively charged species at the isoelectric point for the enzyme (pH 8.7). We have tested this proposal by examining the ionic strength dependence of the association rate constant at various pH values. These data have been interpreted by using the Wherland-Gray equation, which we have shown can be applied to the kinetics of enzyme-coenzyme association. Our results indicate that the shielding of the buffer electrolyte changes from a negative to a positive value as the charge on the protein changes at the isoelectric point. This result is exactly that which is predicted for electrostatic effects that depend on the charge of the protein molecule and is not consistent with predictions based upon the local active site effects. At low ionic strength values of 10 mM or less, approximately 75% of the observed pH dependence results from the enzyme electrostatic effects; the remaining pH dependence may result from active site effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1989

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction.

Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. J., & Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., & Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrostatic diprotein complex yield association constants of greater than or equal to 5 X 10(6) and 1 X 10(5) M-1 at ionic strengths of 1 and 40 mM, respectively, and the rate constant of 1.05 X 10(3) s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.

Animals↗