Intracomplex Electron Transfer Rate Research Articles

Charge-Disproportionation Symmetry Breaking Creates a Heterodimeric Myoglobin Complex with Enhanced Affinity and Rapid Intracomplex Electron Transfer.

We report rapid photoinitiated intracomplex electron transfer (ET) within a "charge-disproportionated" myoglobin (Mb) dimer with greatly enhanced affinity. Two mutually supportive Brownian Dynamics (BD) interface redesign strategies, one a new "heme-filtering" approach, were employed to "break the symmetry" of a Mb homodimer by pairing Mb constructs with complementary highly positive and highly negative net surface charges, introduced through D/E → K and K → E mutations, respectively. BD simulations using a previously developed positive mutant, Mb(+6) = Mb(D44K/D60K/E85K), led to construction of the complementary negative mutant Mb(-6) = Mb(K45E, K63E, K95E). Simulations predict the pair will form a well-defined complex comprising a tight ensemble of conformations with nearly parallel hemes, at a metal-metal distance ∼18-19 Å. Upon expression and X-ray characterization of the partners, BD predictions were verified through ET photocycle measurements enabled by Zn-deuteroporphyrin substitution, forming the [ZnMb(-6), Fe(3+)Mb(+6)] complex. Triplet ET quenching shows charge disproportionation increases the binding constant by no less than ∼5 orders of magnitude relative to wild-type Mb values. All progress curves for charge separation (CS) and charge recombination (CR) are reproduced by a generalized kinetic model for the interprotein ET photocycle. The intracomplex ET rate constants for both CS and CR are increased by over 5 orders of magnitude, and their viscosity independence is indicative of true interprotein ET, rather than dynamic gating as seen in previous studies. The complex displays an unprecedented timecourse for CR of the CS intermediate I. After a laser flash, I forms through photoinduced CS, accumulates to a maximum concentration, then dies away through CR. However, before completely disappearing, I reappears without another flash and reaches a second maximum before disappearing completely.

Probing the Paracoccus denitrificans Cytochrome c1−Cytochrome c552 Interaction by Mutagenesis and Fast Kinetics

Electron transfer (ET) between Paracoccus denitrificans cytochrome (cyt) c(1) and cytochrome c(552) was studied using the soluble redox fragments cyt c(1CF) and cyt c(552F). A new ruthenium cyt c(552F) derivative labeled at C23 (Ru(z)-23-c(552F)) was designed to measure rapid electron transfer with cyt c(1CF) in the physiological direction using flash photolysis. The bimolecular rate constant k(12) decreased rapidly with ionic strength above 40 mM, consistent with a diffusional process guided by long-range electrostatic interactions between the two proteins. However, a new kinetic phase was detected at an ionic strength of <35 mM with the ruthenium photoexcitation technique in which k(12) became very rapid (3 x 10(9) M(-1) s(-1)) and nearly independent of ionic strength, suggesting that the reaction became so fast that it was controlled by short-range diffusion along the protein surfaces guided by hydrophobic interactions. These results are consistent with a two-step model for formation of the final encounter complex. No intracomplex electron transfer between Ru(z)-23-c(552F) and c(1CF) was observed even at the lowest ionic strength, indicating that the dissociation constant of the complex was >30 microM. On the other hand, the ruthenium-labeled yeast cytochrome c derivative Ru(z)-39-Cc formed a tight 1:1 complex with cyt c(1CF) at ionic strengths of <60 mM with an intracomplex electron transfer rate constant of 50000 s(-1). A group of cyt c(1CF) variants in the presumed docking site were generated on the basis of information from the yeast cyt bc(1)-cyt c cocrystal structure. Kinetic analysis of cyt c(1CF) mutants located near the heme crevice provided preliminary identification of the interaction site for cyt c(552F) and suggested that formation of the encounter complex is guided primarily by the overall electrostatic surface potential rather than by defined ions.

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.

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.

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).

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.

Electron transfer from cytochrome c to 8-azido-ATP-modified cytochrome c oxidase.

Bovine heart cytochrome c oxidase (CcO) has been modified by 8-azido-adenosine 5'-triphosphate (8-azido-ATP), and the electron-transfer activity from ferrocytochrome c to the modified CcO under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The kinetics of intermolecular electron transfer between the redox protein partners was shown to be reduced significantly. In addition, there is significant decrease in the binding affinity of the cytochrome c to the oxidase upon 8-azido-ATP modification. The 8-azido-ATP-modified CcO exhibited 50% of the intracomplex electron-transfer rate (ket) and 56% of the association constant (Ka) normally observed between cytochrome c and native CcO under otherwise identical conditions. Since the effective electron transfer rate constant is the product of ket and Ka under nonsaturation conditions, the overall electron-transfer rate has been curtailed by over a factor of 2. Similar observations have been noted with the native CcO in the presence of 3 mM ATP. In contrast, the redox potential of neither CuA nor cytochrome a was altered upon 8-azido-ATP modification or in the presence of 3 mM ATP. Also, no gross structural changes at either the CuA or the cytochrome a site were noted, as evidenced by a lack of any spectral perturbations in the EPR signals from both of these centers. We conclude that ATP modulates the electron transfer from cytochrome c to CcO by interacting with the CcO and altering allosterically the docking. In this manner, ATP can affect the branching of the electron input from ferrocytochrome c to cytochrome a and CuA.

8-Azido-ATP Modification of Cytochrome c: Retardation of Its Electron-Transfer Activity to Cytochrome c Oxidase

Horse heart cytochrome c has been modified by 8-azido-ATP and the electron-transfer activity of the modified cytochrome c's to bovine heart cytochrome c oxidase (CcO) under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The intermolecular electron transfer between the redox protein partners was shown to be extremely slow. The 8-azido-ATP-modified system exhibited less than 5% of the intracomplex electron-transfer rate observed between native cytochrome c and CcO under otherwise identical conditions. The binding affinity of the modified cytochrome c was greatly reduced (3 orders of magnitude) at low ionic strengths; however, it was only slightly reduced (by a factor of 2) relative to the native protein at physiological ionic strengths. Thus, the binding affinity of the ATP-cytochrome c adducts is relatively insensitive to the ionic strength compared to the native enzyme, suggesting that a different docking conformation is assumed by the ATP-cytochrome c adducts in their interaction with the oxidase. Since the redox potential of the modified cytochrome c is close to the value of its native form, we conclude that there has been a change in the docking of the cytochrome c to CcO and the electronic coupling between heme c and CuA upon 8-azido-ATP modification.

Spectroscopic and kinetic characterization of the spinach plastocyanin mutant Tyr83-His: a histidine residue with a high p K value

Tyrosine-83 in spinach plastocyanin (Pc) has been modified by site-directed mutagenesis to a histidine. An NMR titration yields a p K value of 8.44 for this residue. The high value is probably due to the acidic residues close to this site. The reduction potential is increased by 35 mV at pH 7.5, but only slightly, if at all, at pH 8.9. EPR and optical absorption bands associated with the copper site are not affected by the mutation, either at pH 7.5 or at pH 8.9. The electron transfer (ET) to Photosystem I (PS I), as studied by a flash-photolysis technique, is pH dependent for the mutant, being slower than the wild type at pH 7.5 but more similar to it at pH 8.9. The data have been interpreted with a model that includes a rate-limiting conformational change in the Pc-PS I complex which precedes the intracomplex ET (Bottin, H. and Mathis, P. (1985) Biochemistry 24, 6453–6460). The slower kinetics at the lower pH for the mutant is attributed to a dual effect of the protonation of the His-83 residue: (i) A destabilization of the ‘close’ bound conformation, i.e., the one competent in electron transfer, and (ii) a smaller intracomplex ET rate constant, partly due to a smaller driving force for ET. From this it is concluded that the Tyr-83 residue is not a part of the ET pathway to PS I.

Genetic variants in the putidaredoxin-cytochrome P-450cam electron-transfer complex: identification of the residue responsible for redox-state-dependent conformers.

Camphor is hydroxylated in Pseudomonas putida by a three-component system comprised of an oxidase, cytochrome P-450cam, and a two-protein electron-transfer chain, putidaredoxin and putidaredoxin reductase [Tyson et al. (1972) J. Biol. Chem. 274, 5777-5784]. The enzymatic removal of putidaredoxin's C-terminal tryptophan is known to cause a much reduced rate of enzymatic activity in the reconstituted camphor hydroxylase system [Sligar et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3906-3910]. To further study the role of tryptophan in the association and/or electron-transfer reactions of putidaredoxin, the gene coding for the iron-sulfur protein was altered so that the tryptophan codon was either deleted or replaced by Phe, Tyr, Asp, Leu, Val, or Lys. Although the initial evaluation of these variant proteins [Davies et al. (1990) J. Am. Chem. Soc. 112, 7396-7398] showed much reduced velocities of electron transfer between P-450cam and the nonaromatic C-terminal proteins, the relative contributions of the binding specificity and intracomplex electron-transfer rates were not addressed. We report here a complete kinetic characterization of these proteins where the dependence of the rate constant on the putidaredoxin concentration was used to determine the intracomplex electron-transfer rate constants and the association energies for all the putidaredoxins in both oxidation states. The sum of forward and reverse intracomplex electron-transfer rate constants varies from 4.90 s-1 for the Lys C-terminal variant to 172 s-1 for the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Transfer between Cytochrome B5 Surface Mutants and Cytochrome C

Abstract The surface amino acid residues of rat liver Cytochrome b5 (Cyt b5) thought to be involved in forming salt bridges with horse heart Cytochrome c (Cyt c) have been mutated in order to probe the relation between the protein complex structure and the electron transfer function. The intracomplex electron transfer rate constants measured by flash photolysis, in aqueous solution at pH 7.3, range from about 2000–5000 s−1. There is no significant difference between the wild type (WT) and the mutant Cyt b5′s in the electron transfer rate constant. The electron transfer rate constants are also measured under different pH and solvent conditions. These experiments indicate that the protein dynamic fluctuations may play important roles in the search of favorable conformations in order for electron transfer to occur.

Photoinduced electron transfer within complexes between plastocyanin and ruthenium bisbipyridine dicarboxybipyridine cytochrome c derivatives

A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation.

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.

Substituent effects on the electron transfer reactivity of hydroquinones with laccase blue copper

Stopped-flow kinetic studies of the anaerobic reduction of Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) type 1 copper by 25 mono- and disubstituted hydroquinones-(H 2Q-X) have been performed at 25°C and pH 7.0 in 0.5 M phosphate. All of the data are compatible with a mechanism involving rapid enzyme-substrate complex formation followed by rate-limiting intra-complex electron transfer. ES complex formation constants ( Q p ) for many substrates are strikingly insensitive to the electronic characteristics of the substituent X, falling within the range 5–50 M −1. It is shown that this result may be accounted for if only the singly ionized forms of the substituted hydroquinones are bound by the enzyme. All of the substrate exhibiting exceptionally high Q p values ( >50 M −1 ) have X groups capable of functioning as ligands; substituents with lone pairs of electrons may facilitate enzyme-substrate complex formation by enabling hydroquinone to function as a bidentate bridging ligand between the type 2 and type 3 copper sites. Intra-complex electron transfer rate constants for most substrates and remarkably insensitive to the thermodynamic driving force for the oxidation of H 2Q-X to the corresponding semiquinone, the average value for ten substrates being 30 ± 10 s −1 . The electron transfer reactivity of polyphenols with laccase blue copper therefore appears to be controlled largely by protein-dependent activation requirements rather than by the oxidizability of the substrate.