Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.     

Mechanism of electron transfer between myoglobin derivatives and ferricytochrome C

Progress in the studies of the electron transport mechanism in biological systems is greatly hindered by the lack of detailed structural information about the components of these systems. That is why a study of electron transfer between protein molecules with the known spatial organization in model reactions in vitro is of great importance. In this respect the MbO2--Cyt C oxidation-reduction reaction offers unique possibilities. Studies of the effects of pH and ionic strength of the medium on the kinetics of this reaction in combination with chemical modification of single amino acid residues of Mb and Cyt C enabled us to identify those parts of the surface of haemoproteins where the molecules come into "active contact". A variation in the number or/and the arrangement of the charged groups at the "active sites" of the molecules induced by both changing the medium pH and chemical modification of some of these groups lowers markedly the probability of electron transfer in the system (e.g. His GH1 and His A10 in Mb) or blocks it entirely (acylation of Lys 72 (73) or Tyr 74 in Cyt C). Based on the results obtained and on the data of Mb and Cyt C X-ray analysis, the figures of spatial arrangement of the groups at the "active sites" of these molecules are presented.     

Electron transport in hemoproteins. VIII. The effect of chemical modification of ferricytochrome C on the rate of its reduction by oxymyoglobin

The influence of chemical modification of separate amino acid residues in ferricytochrome c (Cyt c) on the rate of the redox reaction with MbO2 has been studied at various pH and ionic strength values. It is shown that alkylation of His-33 and Met-65 by bromacetate does not affect the reaction rate. On the contrary, acylation of Tyr-74 or one of the neighbouring lysines, Lys-72 or Lys-73, by the spin-label N-(2,2',5,5'-tetramethyl-3-carboxypyrrolin-1-oxy)-imidazol diminishes sharply the efficiency of electron transfer in the redox system studied. Besides, unlike the reaction between native proteins, the rate of electron transfer in this case does not depend on ionic strength. The modification of Tyr-74 or Lys 72/43 does not alter the midpoint potential and the entire conformation of Cyt c. The observed effects can therefore be explained by essential disturbance of interactions, first of all, the electrostatic ones in the active complex, which is induced by the attachment of the bulky reagent to the site of "active contact" of Cyt c. Based on the obtained findings and the atomic coordinates of Cyt c, the positions of all charge and some uncharged groups on the surface of Cyt c interacting with myoglobin during electron transfer are presented.     

Controlling the electron transfer rate between oxymyoglobin and ferricytochrome C by alterations in the myoglobin contact site. The role of histidines and electrostatic and steric interactions in the mechanism of the reaction

The kinetics of the redox reaction of sperm whale and pig oxymyoglobins (MbO₂) with ferricytochrome C (CytC) from pig heart has been studied in the pH range 5–8. Also, the effects of histidine (His) modification and of the complexing of both myoglobins with Zn²⁺, on the electron transfer rate, has been investigated. It has been shown that pig MbO₂ reduces Cyt C much more effectively than sperm whale MbO₂. The pH dependence of the reaction rate is shown to result from the influence of two histidines, His 12(A10) and His 119(GH1), in the case of sperm whale myoglobin and only of His GH1 in the case of pig MbO₂. The protonation of His A10 at pH<7.5 decreases the rate of the reaction with Cyt C whereas the ionization of His GH1, on the contrary, increases the electron transfer rate 10–30 times (atI=0.03). The His residues of Cyt C are shown to have no effect on the reaction. Complexing of His GH1 with a zinc ion strongly inhibits the reaction of both sperm whale and pig MbO₂ with Cyt C. The reaction of the zinc-MbO₂ complexes, as distinct from the intact oxymyoglobins, becomes independent of pH and ionic strength. Unlike His A10, His GH1 plays a very important role in the formation of the electron transfer complexes, and is probably directly involved in the charge transfer step. Based on the data obtained, the reactive site of the Mb surface has been identified in the A-GH region. The spatial arrangement of the charged groups in the reactive sites of the two myoglobins has been obtained. The solvent accessibilities of all amino acid residues situated there have been calculated, according to Lee and Richards. In order to explain the different reactivities of sperm whale and pig myoglobins, their electrostatic properties and the steric features of the contact sites have been compared.     

Electron transfer in hemoproteins. VI. The dependence of the reduction rate of ferricytochrome c by oxymyoglobin on ionic strength

The rate of the redox-reaction between MbO2 and ferri-Cyt c has been investigated in the pH range 5-8 under different ionic strength of the solution. The influence of various anions-phosphate, chloride, sulfate and acetat on the rate of the reaction were also studied. It has been shown that under the low ionic strength, I less than 0.1, all pH-dependence curves have pronounced maximum near pH 6.0. While the ionic strength values increase in this interval the reaction rate falls markedly, the profile of lg k versus square root of I/1 + square root of I is linear. Under high ionic strength values, I greater than 0.1, the reaction rate in MbO2-Cyt c system is only slightly influenced by increasing salt concentrations and by pH changing. The results obtained support the idea that the local interactions of charged groups in " active sites" of MbO2 and Cyt c play the most important role in the mechanism of electron transfer. On the contrary net charges of the molecules have a negligible effect on the rate of the reaction. Compared to anions Cl-, SO42- and CH3COO- which influence the reaction rate in an analogous way, phosphate ions have essential inhibiting effect. This is most likely explained by the specific bonding of the phosphate ions to Cty c in the immediate vicinity from the site of the "active contact" with Mb molecule.     

Catalytic effect of ferricyanide on the rate of electron transfer between myoglobin and cytochrome c]

The influence of small amounts of low-molecular electron acceptor, potassium ferricyanide, 1 to 20% relative to the cytohrome c concentration, on the rate of electron transfer in the sperm whale oxymyoglobin--horse heart cytochrome c and deoxymyoglobin--cytochrome c systems (under aerobic and anaerobic conditions, respectively) was studied. At low ionic strength, the redox reaction rate was found to increase proportionally to the concentration of ferricyanide in both redox systems. The effect depends on pH in the pH range 5-8, increasing sharply at pH < 6. It was shown that the enhancing of electron transfer is caused by the complexing of [Fe(CN)6]3- with cytohrome c in the Lys72 region, where one of the two strong binding sites for this anion is determined by NMR. Both the high ionic strength and the chemical modification of Lys72 residue inhibit this effect at low ionic strength, markedly decreasing the rate of reaction with myoglobin. Under the same conditions, the effect of ferricyanide in the reaction of oxy-Mb with yeast cytohrome c, which is isopotential to animal cytochromes c but possesses trimethylated Lys72, was several times smaller. In turn, the chemical modification of His residues in myoglobin and the complexing of zinc ion to His119(GH1) almost completely inhibit electron transfer in the systems. Thus, electron transfer between the proteins must proceed through the formation of the Mb.[Fe(CN)6]3-.Cyt c ternary complex, the contacting sites being localized in the His119(GH1) region of myoglobin and near Lys72 of cytohrome c. The increased electron transfer rate in the presence of [Fe(CN)6]3- can be explained by that its binding near Lys72, firstly, provides better electrostatic interactions in the electron transfer complex and, besides, decreases significantly (about 2-fold) the tunneling distance between the two hemes (two lengths of 1.7 and 1.2 nm instead of one of 2.9 nm).     

Electron transport in hemoproteins. IX. The effect of zinc ions on the rate of oxymyoglobin oxidation by ferricytochrome c

The effect of zink ions, which according to the X-ray data are bound to the His GH1 residue of myoglobin, has been investigated. It is shown that the electron transfer in the system is almost completely inhibited at the equimolar Zn2+ concentration in the pH range 5 to 8. Unlike the reaction between the intact MbO2 and Cyt c, the electron transfer rate in this case does not depend on pH and ionic strength of the solution. Further increase of Zn2+ concentration up to the 20-fold molar excess has no significant effect on the rate of the process. Since the thermodynamic characteristics of the redox reaction between MbO2 and Cyt c are not altered in the presence of Zn2+, the findings obtained can be interpreted as indicating the important role of His GH1 in the formation of productive electron transfer complex.     

Mapping the electron transfer interface between cytochrome b5 and cytochrome c

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.     

Electrostatic effects on modification of charged groups in the active site cleft of subtilisin by protein engineering

The dielectric constant in the active site cleft of subtilisin from Bacillus amyloliquefaciens has been probed by mutating charged residues on the rim and measuring the effect on the pKa value of the active site histidine (His64) by kinetics. Mutation of a negatively charged surface residue, which is 12 to 13 A from His64, to an uncharged one Asp----Ser99) lowers the pKa of the histidine by up to 0.4 unit at low ionic strength (0.005 to 0.01 M). This corresponds to an apparent dielectric constant of about 40 to 50 between Asp99 and His64. The mutation is in an external loop that is known to tolerate a serine at position 99 from homologies with subtilisins from other bacilli. The environment between His64 and Asp99 is predominantly protein. Another charged residue that is at a similar distance from His64 (14 to 15 A) and is also in an external loop that is known to tolerate a serine residue is Glu156, at the opposite side of the active site. There is only water in a direct line between His64 and Glu156. Mutation of Glu----Ser156 also lowers the pKa of His64 by up to 0.4 unit at low ionic strength. This change again corresponds to an apparent dielectric constant of about 40 to 50. The pKa values were determined from the pH dependence of kcat/KM for the hydrolysis of peptide substrates, with a precision of typically +/- 0.02 unit. The following suggests that the changes in pKa are real and not artefacts of experimental conditions: Hill plots of the data for pKa determination have gradients (h) of -1.00(+/- 0.02), showing that there are negligible systematic deviations from theoretical ionization curves involving a monobasic acid: the pH dependence for the hydrolysis of two different substrates (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide and benzoyl-L-valyl-L-glycyl-L-arginyl p-nitroanilide) gives identical results so that the pKa is independent of substrate; the pH dependence is unaffected by changing the concentration of enzyme, so that aggregation is not affecting the results; the shift in pKa is masked by high ionic strength, as expected qualitatively for ionic shielding of electrostatic interactions.     

Mechanism of oxidation of oxymyoglobin by copper ions: comparison of sperm whale, horse, and pig myoglobins

The influence of Cu²⁺ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu²⁺ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is observed in the presence of equimolar Cu²⁺ concentration for sperm whale and horse oxymyoglobins. The curves of pH dependence in both cases are sigmoid with pK _eff corresponding to the ionization. The process is caused by the strong binding of Cu²⁺ to His113 and His116, an analogous His residue being absent in pig Mb. In contrast, rapid oxidation of 10-15% of pig oxy-Mb is observed under the same conditions (fast phase), which is not accompanied by catalysis because the reduced copper is apparently not reoxidized. The complexing of Cu²⁺ with His97 situated near the heme is probably responsible for the fast phase of the reaction. The affinity of His97 for Cu²⁺ must be significantly lower than those of the catalytic His residues since the fast phase does not contribute markedly to the rate of sperm whale and horse oxy-Mb oxidation. Increasing copper concentration does not produce a proportional growth in the oxidation rate of sperm whale and horse oxy-Mbs. Which Cu²⁺ binding sites of Mb make main contributions to the His reaction rate at different Cu²⁺/Mb ratios from 0.25 to 10 is discussed.     

Electrostatic forces in two lysozymes: calculations and measurements of histidine pKa values.

In order to examine the electrostatic forces in globular proteins, pKa values and their ionic strength dependence of His residues of hen egg white lysozyme (HEWL) and human lysozyme (HUML) were measured, and they were compared with those calculated numerically. pKa values of His residues in HEWL, HUML, and short oligopeptides were determined from chemical shift changes of His side chains by 1H-nmr measurements. The associated changes in pKa values in HEWL and HUML were calculated by solving the Poisson-Boltzmann equations numerically for macroscopic dielectric models. The calculated pKa changes and their ionic strength dependence agreed fairly well with the observed ones. The contribution from each residue of each alpha-helix dipole to the pKa values and their ionic strength dependence was analyzed using Green's reciprocity theorem. The results indicate that (1) the pKa of His residues are largely affected by surrounding ionized and polar groups; (2) the ionic strength dependence of the pKa values is determined by the overall charge distributions and their accessibilities to solvent; and (3) alpha-helix dipoles make a significant contribution to the pKa, when the His residue is close to the helix terminus and not fully exposed to the solvent.     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

Role of the hinge protein in the electron transfer between cardiac cytochrome c1 and c. Equilibrium constants and kinetic probes

A role of the hinge protein is studied in the electron transfer reaction between cytochromes c1 and c, using highly purified "one-band" cytochrome c1 and "two-band" cytochrome c1. The results show that the hinge protein (Hp), which is essential for a stable ionic strength-sensitive c1-Hp-c complex, seems to play a certain role in electron transfer between cytochromes c1 and c; Keq for electron transfer reaction between cytochromes c1 and c in the presence of the hinge protein is found to be about 40% higher than that in the absence of the hinge protein at low ionic strength, but no difference exists at high ionic strength. We propose a hypothesis that the hinge protein may function as regulator for the electron transfer reaction between cytochromes c1 and c, and this may be at least one of the roles of the hinge protein in mitochondria.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

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.     

Refolding transition of alpha-chymotrypsin: pH and salt dependence.

It is well known that alpha-chymotrypsin can exist in two major conformational states, only one of which is active. We have examined the pH (pH 2.0--11.0) and salt (ionic strength 0.01--1.0) dependence of the transition between the active and inactive forms in detail. At low pH (pH 2.0--6.0) the equilibrium is very dependent on salt concentration, with high salt concentrations effectively stabilizing the active conformation. This apparent stabilization is an artifact due to the salt-dependent dimerization of alpha-chymotrypsin, and our data show that only active species form dimers and higher aggregates. At neutral pH (6.0--8.0) dimerization is absent, yet an ionic strength dependence remains. The effects show no lyotropic order and appear to be due to preferential salt binding to the active conformation at one or possibly a few sites. Above pH 6 (pH 6.0--11.0), the pH dependence can be described by a two-ionization mechanism at all ionic strengths. We report values for all seven equilibrium constants in the proposed mechanism at four ionic strengths (mu = 0.01, 0.05, 0.2, and 1.0). The transition is the first "refolding" transition to be studied at high precision, but, even so, certain decisions about the mechanism must await higher experimental precision not available with present methods.     

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+.     

Docking and electron transfer studies between rubredoxin and rubredoxin:oxygen oxidoreductase

The interaction and electron transfer (ET) between rubredoxin (Rd) and rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas is studied by molecular modelling techniques. Experimental kinetic assays using recombinant proteins show that the Rd reoxidation by ROO displays a bell-shaped dependence on ionic strength, suggesting a non-trivial electrostatic dependence of the interaction between these two proteins. Rigid docking studies reveal a prevalence for Rd to interact, in a very specific way, with the surface of the ROO dimer near its FMN cofactors. The optimization of the lowest energy complexes, using molecular dynamics simulation, shows a very tight interaction between the surface of the two proteins, with a high probability for Rd residues (but not the iron centre directly) to be in direct contact with the FMN cofactors of ROO. Both electrostatics and van der Waals interactions contribute to the final energy of the complex. In these complexes, the major contributions for complex formation are polar interactions between acidic residues of Rd and basic residues of ROO, plus substantial non-polar interactions between different groups. Important residues for this process are identified. ET estimates (using the Pathways model), in the optimized lowest energy complexes, suggest that these configurations are efficient for transferring electrons. The experimental bell-shaped dependence of kinetics on ionic strength is analysed in view of the molecular modelling results, and hypotheses for the molecular basis of this phenomenon are discussed.     

Nicotinic acetylcholine receptor channel electrostatics determined by diffusion-enhanced luminescence energy transfer

The electrostatic potentials within the pore of the nicotinic acetylcholine receptor (nAChR) were determined using lanthanide-based diffusion-enhanced fluorescence energy transfer experiments. Freely diffusing Tb3+ -chelates of varying charge constituted a set of energy transfer donors to the acceptor, crystal violet, a noncompetitive antagonist of the nAChR. Energy transfer from a neutral Tb3+ -chelate to nAChR-bound crystal violet was reduced 95% relative to the energy transfer to free crystal violet. This result indicated that crystal violet was strongly shielded from solvent when bound to the nAChR. Comparison of energy transfer from positively and negatively charged chelates indicate negative electrostatic potentials of -25 mV in the channel, measured in low ionic strength, and -10 mV measured in physiological ionic strength. Debye-Hückel analyses of potentials determined at various ionic strengths were consistent with 1-2 negative charges within 8 A of the crystal violet binding site. To complement the energy transfer experiments, the influence of pH and ionic strength on the binding of [3H]phencyclidine were determined. The ionic strength dependence of binding affinity was consistent with -3.3 charges within 8 A of the binding site, according to Debye-Hückel analysis. The pH dependence of binding had an apparent pKa of 7.2, a value indicative of a potential near -170 mV if the titratable residues are constituted of aspartates and glutamates. It is concluded that long-range potentials are small and likely contribute little to selectivity or conductance whereas close interactions are more likely to contribute to electrostatic stabilization of ions and binding of noncompetitive antagonists within the channel.