Intracomplex Electron Transfer Research Articles

Mutagenesis of Prochlorothrix Plastocyanin Reveals Additional Features in Photosystem I Interactions

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.

Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome bc1 complex.

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc. The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1. The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.

Formation of a Supramolecular Porphyrin-Spacer-Acceptor Ternary Complex and Intracomplex Electron Transfer

A new ditopic spacer, N-propylisonicotinamidine, has been developed to assemble a ternary supramolecular complex, zinc-tetraphenylporphyrin (ZnTPP)-spacer-acceptor (3,4-dinitrobenzoic acid), via an axial coordination of the spacer to ZnTPP and a salt bridge interaction between the spacer and acceptor. Formation of this ternary complex was probed by 1 H HMR and UV-vis spectroscopy. The ternary complex is formed with an overall association constant of 1.9 x 10 8 M - 2 in dichloromethane. The fluorescence is nearly completely quenched by the electron acceptor in the ternary complex as confirmed by the steady-state fluorescence measurements. The triplet excited state of ZnTPP is also quenched by the electron acceptor in the ternary complex. The ternary complex is also formed in acetonitrile, where fluorescence quenching is observed with an intracomplex quenching rate constant of 1.9 x 10 9 s - 1 . Photoinduced intracomplex electron transfer is invoked as a likely mechanism of the fluorescence quenching and triplet bleaching.

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer.

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.

Microcalorimetric Studies of Rhus vernicifera Laccase Catalytic Oxidation Reactions

The oxidation of substrates with various function groups (i.e., ortho-diphenol, ortho-aminophenol, p-aminophenol, ortho-phenylene diamine and p-phenylene diamine) catalyzed by Laccase have been studied using of LKB-2107 batch microcalorimetry system. The overall reaction enthalpy ΔrHm, Michaelis constant Km, pseudo-first-order rate constant k2 of the reactions and binding energy ΔG0 of Laccase-substrate complex for each substrate have been determined at 298.15 K, pH 7.4. The binding enthalpy ΔH0 and binding entropy ΔS0 of LS complex, when the substrate is Gallic acid and 2,3-dicyan hydroquinone respectively, have been also determined. The rate constants of the reaction between Laccase and these substrates are essentially identical because the rate of Laccase-catalytic reaction is limited by the step of electron transfer from the type 2 Cu(II)-bound substrate to the less exposed type 1 copper site, and the intra-complex electron transfer rate mainly dependents on the amino residues conformational changes. But the rate of the reaction between Laccase and substrates with group -OH is slower than group -NH2, because of the higher energy of bounding electron. The stability of LS complex depends on ΔS0, since ΔH0>0, substrate distinguished by Laccase depends on the value of allosteric entropy of the amino acid residues induced by substrate. The relationship between Laccase and its substrates cannot be regarded as a simple relationship of lock and key, but an induce-fit relationship.

A scanning tunneling microscopy (STM) investigation of complex formation between cytochrome P450cam and putidaredoxin

We have previously reported the scanning tunnelling microscopy (STM) imaging under buffer of the heme monooxygenase cytochrome P450cam from Pseudomonas putida [Faraday Discuss. 116 (2000) 1]. We describe here the adsorption and STM imaging under buffer of complexes of a mutant of cytochrome P450cam, K344C, and wild-type putidaredoxin (Pdx) on gold(111). The images of Pdx on its own on gold(111) are not uniform, presumably due to multiple orientations of protein adsorption because of the presence of five or more cysteines on the protein surface. STM imaging of a 1:1 mixture of P450cam–K344C/Pdx showed a regular array of pairs of different-sized proteins 20–25 Å apart arranged in rows across the gold(111) surface which we attribute to the P450cam/Pdx complex. The images of the pairs are more regular than those of Pdx on its own, probably as a result of complex formation with P450cam partly overcoming the heterogeneity of Pdx adsorption. As far as we are aware this is the first report of STM imaging of a protein/protein complex, and the first direct observation of P450cam/Pdx complex formation which is a key step in the catalytic cycle of P450cam catalysis. The redox centers of the two proteins are ca. 20 Å apart, too far for rapid intracomplex electron transfer. Whether the observed complex is competent for electron transfer or physiologically relevant is not known, and further work is in progress to elucidate the protein–protein interaction.

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides.

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.

Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

Electron transfer properties of iron–sulfur proteins

The details of most electron transfer reactions involving iron–sulfur proteins have remained undisclosed because of the lack of experimental methods suitable to measure precisely the relevant rates. Nuclear magnetic resonance (NMR) provides a powerful means to overcome these problems, at least with selected proteins. A combination of NMR studies and site-directed mutagenesis experiments has been instrumental in defining both the site of interaction and the main trends of the intracomplex electron transfer in the case of rubredoxin electron self-exchange. Analysis of the NMR data obtained for mixtures of different redox levels of several 2[4Fe–4S] ferredoxins provided both first-order, for intramolecular, and second-order, for intermolecular, rate constants. Their dependence as a function of structural changes gave insight into the mechanism of electron transfer in this type of protein. Contrary to some expectations, the high-spin [4Fe–4Se] + clusters assembled in isopotential ferredoxins do not change the intramolecular electron transfer rate as compared to low-spin [4Fe–4S] + homologs. In combination with activity measurements, the kinetic data have been used to model the electron transfer competent complexes between Clostridium pasteurianum ferredoxin and the main enzymes acting as redox partners in vivo.

Definition of the Interaction Domain for Cytochrome con the Cytochrome bc1 Complex: STEADY-STATE AND RAPID KINETIC ANALYSIS OF ELECTRON TRANSFER BETWEEN CYTOCHROME c AND RHODOBACTER SPHAEROIDESCYTOCHROME bc1 SURFACE MUTANTS

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.

Self-assembled complexes of oligopeptides and metalloporphyrins: measurements of the reorganization and electronic interaction energies for photoinduced electron-transfer reactions

Cationic porphyrins form ground state electrostatically associated complexes with anionic oligo-electrolytes such as those formed by a series of glutamic acid (E) residues. Temperature dependencies were measured of the rate constants for intra-complex electron transfer to the triplet state of Pd(II)TMPyP 4+ from a tyrosine (tyr, Y) or tryptophan (trp, W) moiety connected to a glutamic acid tetramer. In complexes such as YE 4, E 2YE 2, YE 4G 10E (G, glycine), and WE 4 these data were used to estimate the reorganization energy (λ) and electronic interaction energy ( H DA) relevant to the process. For all tyr–peptide complexes, λ values were found to be large (λ∼1.60±0.06 eV), reflecting a relatively high medium polarity in the vicinity of tyr residues. It further indicates that the tyr residues in all oligo-peptides are exposed to the aqueous medium in a similar way irrespective of the position of the aromatic moiety in the peptide chain. A significantly lower λ value (λ=1.08 eV) was derived for the tryptophan-containing peptide complex, indicating a relatively higher hydrophobic character of trp compared to tyr. The electronic coupling matrix elements ( H DA) derived for tyr–peptide complexes (5.1 meV for YE 4, 5.4 meV for YE 4G 10E and 7.5 meV for E 2YE 2) were larger than that found for WE 4 (1.1 meV). Molecular dynamics calculations were employed to obtain structural features of the porphyrin–peptide complexes. These showed average distances between the center of mass (COM) of the porphyrin ring and the center of mass of the amino acid aromatic ring of 816±140 pm (YE 4), 800±80 pm (E 2YE 2), 900±130 pm (YE 4G 10E) and 970±160 pm (WE 4). The molecular dynamics calculations were shown to be in good agreement with the experimentally determined electronic interaction energies, strongly suggesting that H DA is primarily responsible for the dependence of the electron-transfer rate constant ( k ET) on the donor–acceptor separation distance and relative orientation. The higher H DA (7.55 meV) derived for tyr incorporated into the middle of the peptide backbone (E 2YE 2) was presumed to be associated with a higher degree of orbital overlap due to a more favorable ring–ring orientation. Overlap parameters (β derived for all peptide–porphyrin complexes were similar (∼0.95±0.06 Å −1), being in good agreement with most literature values for similar systems. Finally, the intra-complex electron-transfer ratio ( k trp/ k tyr) derived from flash photolysis experiments and the corresponding ratio derived from Marcus' theory combined with experimental data from the temperature-dependence investigations and electrochemical measurements were found to be in excellent agreement. This same consistency was found for the couple E 4Y and E 2YE 2. The empirical expression (Moser and Dutton) governing the intraprotein electron-transfer rate constant in native systems combined with our experimental data ( k ET, λ, Δ G 0) yielded tunneling pathway distances in excellent agreement with those arising from the molecular modeling studies. The exception was for the long peptide YE 4G 10E, for which the Quenched Molecular Dynamic (QMD) sampling technique was complicated and is probably inadequate.

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase: II. RAPID KINETIC ANALYSIS OF ELECTRON TRANSFER FROM CYTOCHROMEc TO RHODOBACTER SPHAEROIDES CYTOCHROME OXIDASE SURFACE MUTANTS

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

Role of configurational gating in intracomplex electron transfer from cytochrome c to the radical cation in cytochrome c peroxidase.

Electron transfer within complexes of cytochrome c (Cc) and cytochrome c peroxidase (CcP) was studied to determine whether the reactions are gated by fluctuations in configuration. Electron transfer in the physiological complex of yeast Cc (yCc) and CcP was studied using the Ru-39-Cc derivative, in which the H39C/C102T variant of yeast iso-1-cytochrome c is labeled at the single cysteine residue on the back surface with trisbipyridylruthenium(II). Laser excitation of the 1:1 Ru-39-Cc-CcP compound I complex at low ionic strength results in rapid electron transfer from RuII to heme c FeIII, followed by electron transfer from heme c FeII to the Trp-191 indolyl radical cation with a rate constant keta of 2 x 10(6) s-1 at 20 degrees C. keta is not changed by increasing the viscosity up to 40 cP with glycerol and is independent of temperature. These results suggest that this reaction is not gated by fluctuations in the configuration of the complex, but may represent the elementary electron transfer step. The value of keta is consistent with the efficient pathway for electron transfer in the crystalline yCc-CcP complex, which has a distance of 16 A between the edge of heme c and the Trp-191 indole [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. Electron transfer in the complex of horse Cc (hCc) and CcP was examined using Ru-27-Cc, in which hCc is labeled with trisbipyridylruthenium(II) at Lys-27. Laser excitation of the Ru-27-Cc-CcP complex results in electron transfer from RuII to heme c FeII with a rate constant k1 of 2.3 x 10(7) s-1, followed by oxidation of the Trp-191 indole to a radical cation by RuIII with a rate constant k3 of 7 x 10(6) s-1. The cycle is completed by electron transfer from heme c FeII to the Trp-191 radical cation with a rate constant k4 of 6.1 x 10(4) s-1. The rate constant k4 decreases to 3.4 x 10(3) s-1 as the viscosity is increased to 84 cP, but the rate constants k1 and k3 remain the same. The results are consistent with a gating mechanism in which the Ru-27-Cc-CcP complex undergoes fluctuations between a major state A with the configuration of the hCc-CcP crystalline complex and a minor state B with the configuration of the yCc-CcP complex. The hCc-CcP complex, state A, has an inefficient pathway for electron transfer from heme c to the Trp-191 indolyl radical cation with a distance of 20.5 A and a predicted value of 5 x 10(2) s-1 for k4A. The observed rate constant k4 is thus gated by the rate constant ka for conversion of state A to state B, where the rate of electron transfer k4B is expected to be 2 x 10(6) s-1. The temperature dependence of k4 provides activation parameters that are consistent with the proposed gating mechanism. These studies provide evidence that configurational gating does not control electron transfer in the physiological yCc-CcP complex, but is required in the nonphysiological hCc-CcP complex.

Electron transfer between cytochrome c2 and the tetraheme cytochrome c in Rhodopseudomonas viridis

Kinetics of electron transfer from soluble cytochrome c2 to the tetraheme cytochrome c have been measured in isolated reaction centers and in membrane fragments of the photosynthetic purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. Absorbance changes kinetics in the region of cytochrome α-bands (540–560 nm) were measured at 21 °C under redox conditions where the two high-potential hemes (c-559 and c-556) of the tetraheme cytochrome were chemically reduced. After flash excitation, the heme c-559 donates an electron to the special pair of bacteriochlorophylls and is then re-reduced by heme c-556. The data show that oxidized heme c-556 is subsequently re-reduced by electron transfer from reduced cytochrome c2 present in the solution. The rate of this reaction has a non-linear dependence on the concentration of cytochrome c2, suggesting a (minimal) two-step mechanism involving the f ormation of a complex between cytochrome c2 and the reaction center, followed by intracomplex electron transfer. To explain the monophasic character of the reaction kinetics, we propose a collisional mechanism where the lifetime of the temporary complex is short compared to electron transfer. The limit of the halftime of the bimolecular process when extrapolated to high concentrations of cytochrome c2 is 60 ± 20 μs. There is a large ionic strength effect on the kinetics of electron transfer from cytochrome c2 to heme c-556. The pseudofirst-order rate constant decreases from 1.1 × 107 M-1 s-1 to 1.3 × 106 M-1 s-1 when the ionic strength is increased from 1 to 1000 mM. The maximum rate (1.1 × 107 M-1 s-1) was obtained at about 1 mM ionic strength. This dependence of the rate on ionic strength s uggests that attractive electrostatic interactions contribute to the binding of cytochrome c2 with the tetraheme cytochrome. On the basis of our data and of previous molecular modelling, it is proposed that cytochrome c2 docks close to the low-potential heme c-554 and reduces heme c-556 via c-554.

Binding and Electron Transfer between Cytochrome b5 and the Hemoglobin α- and β-Subunits through the Use of [Zn, Fe] Hybrids

We have measured the binding affinity (KA) and electron transfer (ET) rate constants (k) for the complex of hemoglobin (Hb) and cytochrome b5 (b5), using triplet quenching titrations of mixed-metal [ZnM, Fe3+(N3-)] Hb hybrids and of fully substituted Zn−mesoporphyrin (ZnM)Hb by b5 (trypsin-solubilized, bovine) (pH values 6.0 and 7.0). The use of the mixed-metal Hb hybrids with Zn in one chain type allows us to selectively monitor the 3ZnP → Fe3+P ET reaction of Fe3+b5 with either the α-chains or the β-chains. The self-consistent analysis of the results for the mixed-metal hybrids and those for the (ZnM)Hb allows us to determine the reactivity and affinity constants for the interactions of b5 with the individual subunits of T-state Hb. The results confirm that ET occurs within a complex between b5 and Hb, not through a simple bimolecular collision process. At pH 6.0, the binding affinity constant of the α-chains (Kα ≈ 2.0 × 104 M-1) is ∼4-fold larger than that of the β-chains (Kβ = 4.9 × 103 M-1); the intracomplex ET rate constant of the α-chains (kα ≈ 1500 s-1) is ∼2-fold larger than that of the β-chains (kβ ≈ 850 s-1). The binding affinity and ET rate constant of the α-chains both decrease as the pH is increased from 6.0 to 7.0; the binding affinity of the β-chains is essentially the same at pH 6.0 and 7.0, while the ET reactivity decreases. The kinetic results are consistent with a docking model in which each subunit binds a molecule of b5. However, they permit an alternative in which b5 reacts with the α-chains by binding at a site which spans the α1β2 dimer interface.

Stereoselective Electron-Transfer Reaction between Metmyoglobin and Chiral Viologen-Radical Cation through Pre-Complexation

Abstract Metmyoglobin was reduced by chiral viologen-radical cations containing ((1-phenyl- or ((1-cyclohexylethyl)carbamoyl)-methyl group through pre-complexation followed by intracomplex electron transfer; (S,S)-isomers of chiral viologens showed slightly higher association constants as well as larger intracomplex electron-transfer rates.

Effects of Temperature and Δ G° on Electron Transfer from Cytochrome c2 to the Photosynthetic Reaction Center of the Purple Bacterium Rhodobacter sphaeroides

The kinetics of electron transfer from cytochrome c 2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy. Rereduction of P + induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P +/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type). Rate constants for first-order electron donation within preformed reaction center–cytochrome c 2 complexes and for the bimolecular oxidation of free cytochrome c 2 have been obtained by multiexponential deconvolution of the kinetics. At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force −Δ G° is varied over a range of 350 meV. The temperature and Δ G° dependences of the rate constant fit the Marcus equation well. Global analysis yields a reorganization energy λ = 0.96 ± 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10 −4 eV to 2.5 10 −4 eV. Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than ∼200 cm −1. An additional slower kinetic component of P + reduction, attributed to electron transfer from cyt c 2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon Δ G°, characterized by a similar value of λ (0.8 ± 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude. In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K. The free energy dependence of the second-order rate constant for oxidation of cyt c 2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated.

Electrostatic effects on electron-transfer kinetics in the cytochrome f-plastocyanin complex

In a complex of two electron-transfer proteins, their redox potentials can be shifted due to changes in the dielectric surroundings and the electrostatic potentials at each center caused by the charged residues of the partner. These effects are dependent on the geometry of the complex. Three different docking configurations (DCs) for intracomplex electron transfer between cytochrome f and plastocyanin were studied, defined by 1) close contact of the positively charged region of cytochrome f and the negatively charged regions of plastocyanin (DC1) and by (2, 3) close contact of the surface regions adjacent to the Fe and Cu redox centers (DC2 and DC3). The equilibrium energetics for electron transfer in DC1-DC3 are the same within approximately +/-0.1 kT. The lower reorganization energy for DC2 results in a slightly lower activation energy for this complex compared with DC1 and DC3. The long heme-copper distance (approximately 24 A) in the DC1 complex drastically decreases electronic coupling and makes this complex much less favorable for electron transfer than DC2 or DC3. DC1-like complexes can only serve as docking intermediates in the pathway toward formation of an electron-transfer-competent complex. Elimination of the four positive charges arising from the lysine residues in the positive patch of cytochrome f, as accomplished by mutagenesis, exerts a negligible effect (approximately 3 mV) on the redox potential difference between cyt f and PC.

Photoinduced electron-transfer reaction between cytochrome c and zinc myoglobin modified with diethylenetriaminepentaacetic acid

The kinetics and mechanism of the photoinduced electron-transfer reaction between cytochrome c and zinc-substituted myoglobin, the latter of which lysine residue was modified with diethylenetriaminepentaacetic acid, were studied in an aqueous solution at 25°C, pH = 7.0 (a 10 mM phosphate buffer) and ionic strengths from 0.02 to 0.32 M (NaCl). The excited triplet-state of the modified zinc myoglobin was efficiently quenched by the oxidized cytochrome c within a diprotein complex. Both the intracomplex electron-transfer quenching rate constant and the binding constant of the diprotein complex decreased with increasing ionic strengths.

Cross-Linked Electron Transfer Complex between Cytochrome c2 and the Photosynthetic Reaction Center of Rhodobacter sphaeroides

Electron donation from the soluble cytochrome (cyt) c2 to the photooxidized primary donor, P+, of reaction centers isolated from Rhodobacter sphaeroides was studied by using chemical zero-length cross-linking. This cross-linking stabilizes a 1:1 covalent complex between subunit M of the reaction center and cyt c2. In 80% of the reaction centers, P+ generated by a laser flash is reduced by covalently bound cyt c2. Kinetics of P+ reduction show (i) a fast phase with a half-life of 0.7 micros similar to that observed for electron transfer in the noncovalent proximal complex and (ii) a slow phase (t1/2 = 60 micros) that is attributed to a cyt c2 bound less favorably for electron transfer. Its relationship with similar kinetic phases attributed to a distal conformation of the complex in previous studies is discussed. Both kinetic phases are slightly accelerated upon addition of glycerol. Upon addition of reduced soluble cyt c2 to the cross-linked complex the kinetics of both phases are not affected. The kinetics of P+ reduction following the second flash (20 ms after the first) show that a complex is formed between soluble cyt c2 and the cross-linked complex, in which electron transfer takes place in the millisecond time domain. Cross-linked cyt c2 in complexes which give rise to the two kinetic phases of P+ reduction shows almost pH-independent midpoint redox potentials between pH 6 and 9.5. This behavior is at variance with that of free cyt c2, the midpoint potential of which is affected by at least two protonable groups within this pH range. The cross-linked RC-cyt c2 complex allowed study of the effects of temperature on the electron transfer reaction without a possible disturbance by dissociation of the complex. In the 250-300 K range, Arrhenius behavior is observed showing activation energies of 11.7 and 8.0 kJ/mol for the faster and the slower kinetic phases, respectively, which are remarkably lower than the activation energy of 20.5 kJ/mol for the fast P+ reduction by soluble cyt c2 [Venturoli, G., Mallardi, A., & Mathis, P. (1993) Biochemistry 32, 13245-13253]. Between 250 and 230 K, a fall-off in amplitude is observed for both kinetic phases indicating that intracomplex electron transfer is blocked at low temperatures.