Electron Self-exchange Rate Research Articles

Reduction of Escherichia coli ribonucleotide reductase subunit R2 with eight water-soluble ferrocene derivatives

Water soluble ferrocenes [Fe(Cp)(CpL)], where Cp− is the η5-cyclopentadienide ligand and the side chain L is (a) the carboxylic acid group –(CH2)xCO2H with x=0–4 (I–V); (b) the complex x=2 with the β-methylene mono-methyl substituted (VI); (c) the amine hydrochloride derivative with L=CH(Me) NH3+ (VII); and (d) the complex with two Cp rings bridged by the amine hydrochloride –CH(NH3+)CH2CH2− (VIII); have been prepared, and are used as one-equivalent reductants for the active-R2 subunit of Escherichia coli ribonucleotide reductase. Formal reduction potentials E1°′ (25°C) of the carboxylates of acids I–VI in 20 mM NaOH, and of the amine hydrochlorides VII and VIII in water were determined by cyclic voltammetry, and are in the range 0.308–0.550 V versus nhe, I=0.100 M (NaCl). Second-order rate constants k12 (25°C) for the reduction of active-R2 were determined by UV–Vis spectrophotometry, and are in the range 0.15–0.50 M−1 s−1 at I=0.100 M. A free-energy plot of logk12 versus E°′ values gives no clearcut unidirectional trend. Since from present information the electron self-exchange rate constant for the [Fe(Cp)2]+/[Fe(Cp)2] couple is favourable (>7×106 M−1 s−1 in methanol at 25°C), it would appear that electron-transfer from the ferrocenes via Trp-48, Asp-237, His-118 to the FeIII2 site on R2 is much slower than expected, and smaller than with the organic radical reductants previously studied. Electron-transfer from some other position on the protein surface to the Tyr is considered as an alternative.

Significant enhancement of electron transfer reduction of NAD(+) analogues by complexation with scandium ion and the detection of the radical intermediate-scandium ion complex.

4-Acetyl-N,N-diisopropyl-1-benzylnicotinamidinium ion (ABNA(+)) and 1-benzyl-4-phenylnicotinamidinium ion (PhBNA(+)) were newly synthesized as NAD(+) analogues to examine the electron-transfer reactivity and the effects of metal ions on the reactivity in comparison with those of 1-benzylnicotinamidinium ion (BNA(+)) and 1-methyl-4-phenylpyridinium ion (MPP(+)) which has no amide or acetyl group. A remarkable positive shift in the one-electron reduction potential of ABNA(+) was observed in the presence of Sc(3+) which forms a 1:1 complex with ABNA(+) through both acetyl and amide groups, whereas no such shift in the presence of Sc(3+) was observed for the one-electron reduction of MPP(+) which has no acetyl or amide group. Similar but less positive shifts in the one-electron reduction potentials were observed in the presence of Sc(3+) for the one-electron reduction of BNA(+) and PhBNA(+) both of which have only one amide group. The rate of electron-transfer reduction of ABNA(+) is enhanced significantly by the complexation with Sc(3+) to produce stable ABNA(*)-Sc(3+) complex which has been successfully detected by ESR. The electron self-exchange rates of the MPP(*)/MPP(+) system have been determined from the ESR line width variation and are compared with those of the ABNA(*)/ABNA(+) system.

Magnetic field effects on the pyrene—dicyanobenzene system: determination of electron self-exchange rates by MARY spectroscopy

An experimental determination has been made of electron self-exchange rates between the radical anions of 1,2-, 1,3- and 1,4-dicyanobenzene (DCB) and the respective neutral molecules applying steady-state field-modulated MARY (magnetic field effect on reaction yield) spectroscopy. For the first time this has been achieved successfully for compounds whose self-exchange rate constants can be obtained independently by alternative methods such as EPR linebroadening. In this study, pyrene was used as an electron donor to generate the spin-correlated radical ion pair (pyrene·+ DCB·−) essential for MARY spectroscopy. The radical ion pair is in equilibrium with an exciplex whose magnetic field affected fluorescence was recorded as a function of the magnetic field to yield the MARY spectrum. Due to lifetime uncertainty energy broadening of spin levels caused by electron self-exchange, the characteristic B 1/2 value increases with the concentration of DCB in the sample. The rate constant of self-exchange was obtained from the slope of the linear part in the plot of B1/2 versus DCB concentration. The values range between 6 x 108 M−1 s−1 and 1.4 x 1010 M−1 s−1, depending on the DCB isomer and solvent. Comparison with literature data from EPR linebroadening measurements shows good agreement.

A general method for determining the electron self-exchange rates of blue copper proteins by longitudinal NMR relaxation.

A general NMR method is presented that allows a precise determination of the second-order rate constant, k(ese), for the electron self-exchange in blue copper proteins, from the longitudinal relaxation rates of the nuclei in the protein. The method relies on the use of partly oxidized (paramagnetic) samples of the protein. In contrast to previous NMR approaches for the determination of electron self-exchange rates, the applicability of the method extends beyond the slow-exchange limit, k(ese)c << R(ip), i = 1, 2, where c is the protein concentration, and R(ip) is the paramagnetic relaxation enhancement of the observed nuclei.

Determination of electron self-exchange rate constants with MARY spectroscopy: Dependence on the fluorophore

The influence of electron self-exchange on the magnetic field effect of various exciplex–radical ion pair (RIP) systems (1,2- and 1,4-dicyanobenzene in combination with the fluorophores pyrene, N-methylcarbazole and N-ethylcarbazole in tetrahydrofurane) has been investigated using magnetic-field-effect-on-reaction-yield (MARY) spectroscopy. By varying the concentration of the diamagnetic counterpart of one of the radicals in the RIP, the dependence of the linewidth on the electron self-exchange rate has been studied. The functional behaviour of the linewidth-related parameter B1/2 with respect to the molecule concentration is governed by two opposing effects: lifetime uncertainty broadening of spin levels leads to a linear increase at low concentrations, whereas weakened hyperfine coupling of the corresponding radical ion at high concentrations causes B1/2 to decrease again until a saturation value is reached. From the linear relationship at low concentrations an estimate of the self-exchange rate constant is obtained.

Solvent dynamical effects on the electron self-exchange rate of the TEMPO˙/TEMPO+couple (TEMPO = 2,2,6,6-tetramethyl-1-piperidinyloxy radical) : Part I. ESR-linebroadening measurements at T = 298 K

Rate constants of the electron self-exchange for the TEMPO˙/TEMPO+ couple were measured in acetonitrile, benzonitrile, deuterium oxide, propylene carbonate, THF and water at T = 298 K. The experimental rate constants cover a range of 0.88 × 108 ⩽ ket ⩽ 5.0 × 108 M−1 s−1 and clearly show a solvent dynamical “friction” effect controlled by the longitudinal relaxation time of the solvent τL.

Application of Marcus cross-relation to mixed inorganic–organic redox couples. A stopped-flow study of the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine with various oxidantsIn memory of Lennart Eberson.

The rate constants of the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPPD) are measured in water by means of stopped-flow techniques using different inorganic ions, like Fe(CN)63−, MnO4−, Co(NH3)63+, Ru(OH)3+6. The rates do not depend on the different oxidation potentials, but can be correlated well with calculated rate constants from the Marcus cross-relation. The electron self-exchange rates of the inorganic oxidants range over nine orders of magnitude. Measurements were performed at T = 293 K within a pH-range of 7–9.

Electron Transfer Dynamics in Molten Salts of Mono- and Dinuclear Ruthenium Complexes

We have synthesized highly viscous, room-temperature, molten salts by associating various Ru(NH3)5L cations (L = 4,4‘-bipyridine, pyrazine, pyridine, 3-chloropyridine, benzonitrile) with polyether-tailed sulfonate anions. Microelectrode voltammetry in the undiluted melts yields, on the basis of charge transport occurring by electron hopping, electron self-exchange rate constants (kEX) for the various Ru3+/2+ couples. The rate constant (and activation barrier) for the pyrazine bridged binuclear pentaamineruthenium melt (Creutz-Taube ion) is similar to those obtained for mononuclear pentaammine[ligand]ruthenium melts, meaning that charge transport in the former is dominated by the rate of intermolecular, not intramolecular, electron transfer. The data at 35 °C are (Creutz−Taube ion) kEX = 3.7 × 104 M-1 s-1, (pyridine) 1.1 × 104 M-1 s-1, (3-chloropyridine) 2 × 104 M-1 s-1, and (benzonitrile) 3 × 104 M-1 s-1. All kEX values are smaller than those for [Ru2+/3+(bpy)3] in semisolid melts having equivalent MePEG ...

Quantum mechanical investigation of the inner-sphere reorganization energy of cyclooctatetraene/cyclooctatetraene radical anion. Part I

The inner-sphere reorganization energy of the electron self-exchange of the couple cyclooctatetraene/cyclooctatetraene radical anion has been investigated by quantum mechanical calculations. The more stable Jahn Teller distorted B 2 g conformation of the radical anion has been used in this study. Two different theories have been applied in this first part. The harmonic approximation in the classical Marcus scheme has been modified by using projected force constants, which are obtained from the complete force constant matrix and the geometry changes of the molecule during the ET (introduced by Mikkelsen). A different approach (introduced by Nelsen) combines the different energies of the neutral and radical anion with and without relaxation corresponding to the vertical ionization potential and the vertical electron affinity. The electronic energies of the neutral molecule and the radical anion differ dramatically applying three different levels of quantum mechanical calculations (UAM1, UB3LYP, PMP2 with three different basis sets with and without diffuse functions). Nevertheless the Nelsen method gives almost consistent results for the inner-sphere reorganization energies: 120.1 kJ/mol for semiempirical UAM1 method, 159.3 kJ/mol, 156.4 kJ/mol and 158.3 kJ/mol for density functional UB3LYP/6–31G*, UB3LYP/6–31++G* and UB3LYP/AUG–cc–pVDZ calculations and 192.5 kJ/mol for ab-initio PMP2/6–31G* investigations, respectively. These values are in agreement with earlier experimental work supposing the total reorganization energy to be larger than 38 kcal/mol assuming an electron self-exchange rate of 10 4 M −1s −1. The simple harmonic approximation of Marcus relation has not yet been applied for a molecule like cyclooctatetraene with large torsional geometry changes. Using the projected force constants after scaling, considerably different results for the inner-sphere reorganization energy have been calculated: 738.1 kJ/mol for the UB3LYP/6–31G*, 743.3 kJ/mol for UB3LYP/6–31++G* and 759.1 kJ/mol for UB3LYP/AUG–cc–pVDZ level of theory. Comparison with our concentration dependent EPR experiments are controversial to the earlier experimental results, but the latter supports the assumption that the electron self-exchange occurs in a time scale so that the molecules cannot complete their vibrational motions. Therefore the projected Marcus relation is not valid for cyclooctatetraene/cyclooctatetraene radical anion including a large torsional change during the electron transfer.

Electron-transfer kinetics of tris(2-(methylthioethyl))aminecopper(II/I). A tripodal ligand complex exhibiting virtual C3v symmetry.

The tripodal ligand TMMEA (tris(2-methylthioethyl)amine) forms a trigonal bipyramidal complex with copper(II) in which the bridgehead nitrogen occupies one axial site, a solvent molecule (or anion) occupies the opposite axial site, and the three thioether sulfurs occupy the three planar sites. Upon reduction to copper(I), the axial solvent molecule (or anion) dissociates to leave a trigonal pyramidal complex with shortened Cu-S bonds and an elongated Cu-N bond. Therefore, both oxidation states maintain virtual C3v symmetry similar to that found in the type 1 blue copper protein sites. The electron-transfer cross-reaction rate constants have been determined for the Cu(II/I)(TMMEA) system reacting with three reductants and three oxidants. The Marcus cross relation was then utilized to generate apparent values for the Cu(II/I) electron self-exchange rate constant (k(11)) from the kinetic data for each of the six reactions. The median value obtained from the three reduction reactions is log k(11(Red)) = -1.5 while the median from the three oxidation reactions is log k(11(Ox)) = +0.9. This difference of 2.4 orders of magnitude is consistent with the dual-pathway square scheme mechanism which we have previously proposed for electron transfer in Cu(II/I) complexes. For this tripodal ligand system, however, the pathway involving a metastable Cu(II)L intermediate (pathway B) appears to be preferred over the pathway involving a metastable Cu(I)L intermediate (pathway A), which is opposite to the trend we have previously observed for a number of systems involving macrocyclic and acyclic tetrathiaethers. Both pathways exhibit relatively sluggish electron-transfer kinetics which is attributed to the rupture/formation of the strongly bound inner-sphere water molecule and the accompanying solvent reorganization.

High sensitivity of the fluorine NMR signals of difluorovinyl analogs of natural hemin: reconstituted heme proteins and self‐exchange electron transfer in model compounds

AbstractThe chemical shifts of fluorine in difluorovinyl deuteroporphyrin iron complexes were shown to be very sensitive to the spin state of the metal and the nature of the ligand(s). Reconstituted myoglobin was used as a model heme protein with an exchangeable heme. Large variations in the fluorine chemical shifts in both the ferric and ferrous states were observed. This strong sensitivity to the nature of the metal ligand and the structural resemblance to natural hemin make this fluorinated porphyrin a good probe for the study of heme proteins. The large variations of chemical shifts depending on the oxidation state also permitted the measurement of the electron self‐exchange rate constants of bis(1‐methylimidazole)iron complexes in various solvents by analysis of line broadening of the 19F NMR signals. The experimental rate constants were strongly affected by the nature of the solvent, varying from 3.9 × 107 to 24.1 × 108 mol l−1 s−1 for DMSO‐d6 and acetone‐d6, respectively. The solvent parameters were used to estimate the outer‐sphere reorganization energies. The experimental rate constants in chloroform and in DMSO are in good agreement with these calculated outer‐sphere reorganization energies. Copyright © 2001 John Wiley &amp; Sons, Ltd.

Effects of dimerization on protein electron transfer.

In order to investigate the relationship between the rate of protein-protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a site for covalent cross-linking was engineered by replacing the surface-exposed asparagine 42 with a cysteine. This mutation enabled the formation of disulfide-linked homo-dimers of azurin. Based on NMR line-broadening experiments, the electron self-exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7) x 10(5)M(-1)s(-1), which is a seven-fold decrease relative to wild-type azurin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intramolecular electron transfer within a dimer and intermolecular electron transfer between two dimers, the e.s.e. rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag-Cu)-N42C dimers. As Zn and Ag are redox inactive, the intramolecular electron transfer reaction in these latter dimers can be eliminated. The e.s.e. rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determined. This rate is compatible with a Cu-Cu distance of 18 A or more, which is larger than the Cu - Cu distance of 15 A observed in the wild-type crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu-Cu distance should be in the range of 17 A < rCu-Cu < 28 A, which is in agreement with the experimental findings. For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation. Direct cross-linking may disturb the formation of such an optimal electron transfer complex.

Direct evidence for a geometrically constrained "entatic state" effect on copper(II/I) electron-transfer kinetics as manifested in metastable intermediates.

The absolute magnitude of an "entatic" (constrained) state effect has never been quantitatively demonstrated. In the current study, we have examined the electron-transfer kinetics for five closely related copper(II/I) complexes formed with all possible diastereomers of [14]aneS(4) (1,4,8,11-tetrathiacyclotetradecane) in which both ethylene bridges have been replaced by cis- or trans-1,2-cyclohexane. The crystal structures of all five Cu(II) complexes and a representative Cu(I) complex have been established by X-ray diffraction. For each complex, the cross-reaction rate constants have been determined with six different oxidants and reductants in aqueous solution at 25 degrees C, mu = 0.10 M. The value of the electron self-exchange rate constant (k(11)) has then been calculated from each cross reaction rate constant using the Marcus cross relation. All five Cu(II/I) systems show evidence of a dual-pathway square scheme mechanism for which the two individual k(11) values have been evaluated. In combination with similar values previously determined for the parent complex, Cu(II/I)([14]aneS(4)), and corresponding complexes with the two related monocyclohexanediyl derivatives, we now have evaluated a total of 16 self-exchange rate constants which span nearly 6 orders of magnitude for these 8 closely related Cu(II/I) systems. Application of the stability constants for the formation of the corresponding 16 metastable intermediates--as previously determined by rapid-scan cyclic voltammetry--makes it possible to calculate the specific electron self-exchange rate constants representing the reaction of each of the strained intermediate species exchanging electrons with their stable redox partners--the first time that calculations of this type have been possible. All but three of these 16 specific self-exchange rate constants fall within--or very close to--the range of 10(5)-10(6) M(-1) s(-1), values which are characteristic of the most labile Cu(II/I) systems previously reported, including the blue copper proteins. The results of the current investigation provide the first unequivocal demonstration of the efficacy of the entatic state concept as applied to Cu(II/I) systems.

Electron self-exchange kinetics in two average-valence dicopper cryptates.

Electron self-exchange rates, reported for two average-valence dicopper cryptates are, at around 10(5) M-1 s-1, at the high end of the range for small-molecule model compounds. The cross-exchange reaction rate, and those for oxidation with [Co(ox)3]3-, are consistent with an outer-sphere reaction mechanism. Despite the necessity for copper-copper bond making and breaking in the course of redox for one of the cryptates, the self-exchange rate is not decreased relative to the other cryptate, showing that this step is not rate limiting.

Electron self-exchange in the solid-state: cocrystals of hydroquinone and bipyridyl triazole.

Solid-state voltammetry, spectroscopy, and microscopy studies have been used to probe the proton and electron conductivity within a self-assembled cocrystal, HQBpt. This crystallographically defined material contains 3,5-bis(pyridin-2-yl)-1,2,4-triazole, HBpt, dimers that are pi-stacked and hydrogen bonded to 1,4-hydroquinone, H(2)Q, in a herringbone arrangement. When deposited onto platinum microelectrodes, the cocrystal exhibits a well-defined voltammetric response corresponding to oxidation of H(2)Q to the quinone, Q, across a wide range of voltammetric time scales, electrolyte compositions, and pH values. Scanning electron microscopy reveals that redox cycling in aqueous perchlorate solutions in which the pH is systematically varied from 1 to 7 triggers electrocrystallization and the extensive formation of rodlike crystals. Fast scan rate voltammetry reveals that the homogeneous charge transport diffusion coefficient, D(app), is independent of the perchlorate concentration for 0.1 < [ClO(4)(-)] < 1.0 M (pH 6.6) at 3.14 +/- 0.11 x 10(-)(9) cm(2) s(-)(1). Moreover, D(app) is independent of the perchloric acid concentration for concentrations greater than approximately 2.0 M, maintaining a value of 4.81 +/- 0.07 x 10(-)(8) cm(2) s(-)(1). The observation that D(app) is independent of the supporting electrolyte suggests that the rate-determining step for homogeneous charge transport is not the availability of charge-compensating counterions or protons, but the dynamics of electron self-exchange between H(2)Q and Q. We have used the Dahms-Ruff formalism to determine electron self-exchange rate constants which are 2.84 +/- 0.22 x 10(9) and 9.69 +/- 0.73 x 10(10) M(-)(1) s(-)(1) for pH values greater than approximately 2.0 and less than -0.3, respectively. Significantly, these values are more than 2 orders of magnitude larger that those found for benzoquinone self-exchange reactions in aqueous solution. These results indicate that hydrogen bonds play an important role in supporting rapid electron transfer. The increase in D(app) between pH 1.0 and -0.3 is associated with protonation of the HBpt moieties, which triggers a reversible change in the material's structure.

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy.

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D 1H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D 1H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R2p of the resonances can be determined, and the conditions k << R2p or delta omega(p) >> k >> R2p are fulfilled, delta omega(p) being the frequency separation between corresponding diamagnetic and paramagnetic sites.

Electron self-exchange reaction of viologen and its derivatives in poly(ethylene glycol)

The electron self-exchange rates ( k ex) of viologen and its derivatives are estimated by using microelectrode voltammetry in poly(ethylene glycol) films. The dependences of supporting electrolyte concentration and sizes of viologen and its derivatives on k ex and diffusion coefficients ( D) are discussed. Results show that k ex increases with the decrease of supporting electrolyte concentration and sizes of reactants.

The Met99Gln mutant of amicyanin from Paracoccus versutus.

The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus. Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement. M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin. In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin. For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M. These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin. A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.

Effect of conformational constraints on gated electron-transfer kinetics. 3. Copper(II/I) complexes with cis- and trans-cyclopentanediyl-1,4,8,11-tetrathiacyclotetradecane.

Previous kinetic and electrochemical studies of copper complexes with macrocyclic tetrathiaethers-such as 1,4,8,11-tetrathiacyclotetradecane ([14]aneS4)-have indicated that electron transfer and the accompanying conformational change occur sequentially to give rise to a dual-pathway mechanism. Under appropriate conditions, the conformational change itself may become rate-limiting, a condition known as "gated" electron transfer. We have recently hypothesized that the controlling conformational change involves inversion of two donor atoms, which suggests that "gated" behavior should be affected by appropriate steric constraints. In the current work, two derivatives of [14]aneS4 have been synthesized in which one of the ethylene bridges has been replaced by either cis- or trans-1,2-cyclopentane. The resulting copper systems have been characterized in terms of their Cu(II/I)L potentials, the stabilities of their oxidized and reduced complexes, and their crystal structures. The electron self-exchange rate constants have been determined both by NMR line-broadening and by kinetic measurements of their rates of reduction and oxidation with six or seven counter reagents. All studies have been carried out at 25 degrees C, mu = 0.10 M (NaClO4 and/or Cu(ClO4)2), in aqueous solution. Both Cu(II/I) systems show evidence of a dual-pathway mechanism, and the electron self-exchange rate constants representative of both mechanistic pathways have been determined. The first-order rate constant for gated behavior has also been resolved for the Cu(I)(trans-cyclopentane-[14]aneS4) complex, but only a limiting value can be established for the corresponding cis-cyclopentane system. The rate constants for both systems investigated in this work are compared to values previously determined for the Cu(II/I) systems with the parent [14]aneS4 macrocycle and its derivatives involving phenylene and cis- or trans-cyclohexane substituents. The results are discussed in terms of the influence of the fused rings on the probable conformational changes accompanying the electron-transfer process.

Loop-Directed Mutagenesis of the Blue Copper Protein Amicyanin fromParacoccus versutusand Its Effect on the Structure and the Activity of the Type-1 Copper Site

Four loop-mutants of the blue copper protein amicyanin from Paracoccus versutus have been constructed and characterized. The mutations replaced the loop containing three (Cys93, His96, Met99) of the four copper ligands in amicyanin by the “ligand loops” of P. aeruginosa azurin (AmiAzu), A. faecalis pseudoazurin (AmiPaz), P. nigra plastocyanin (AmiPcy), and P. aureofaciens nitrite reductase (AmiNiR). The copper centers of all variants appear to be perfect type-1 Cu sites although the AmiNiR variant exhibits diminished stability. The optical spectra of the AmiAzu and AmiPaz variants display a significant dependence on temperature. Excitation at 457 nm as well as 647 nm results in similar resonance Raman spectra. The reduction potentials of the three stable variants are all higher than that of wt amicyanin. The reduced forms of the loop-mutants protonate at the C-terminal histidine, with pKa values of 5.6 (AmiAzu), 5.4 (AmiPaz), and 5.7 (AmiPcy) (6.8 for wt amicyanin). AmiAzu is the first known cupredoxin with more than two amino acids between the cysteine and histidine ligands that undergoes this protonation. The electron self-exchange rate constants at 25 °C are 5.7−7.5 times lower for the loop mutants than for wt amicyanin (1.2 × 105 M-1 s-1). The results are interpreted by taking into consideration that three metal ligands are intimately connected with the stable β-sandwich structure of the cupredoxin. This leaves considerable freedom in positioning the fourth one, the C-terminal His, on the “ligand loop”. This explains the ease by which the C-terminal histidine ligand can be excised and replaced by external ligands without losing the metal binding property of the protein. The results also help understand the remarkable evolutionary success of the combination of cupredoxin fold and Cu site for mediating biological electron transfer.