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Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k(H)/k(D), is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (-56.5 J K(-1) mol(-1)) and in deuterium oxide (-35.7 J K(-1) mol(-1)). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential (E(0')) of Cu(2+/+) at 298 K is 10 mV more positive in D(2)O than in H(2)O. The temperature dependence of E(0') is also different, yielding entropy changes of -57 J K(-1) mol(-1) in water and -84 J K(-1) mol(-1) in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to DeltaS from the temperature dependence of E(0') is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H(2)O than in D(2)O (0.001 nm K(-1)) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E(0'). Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.

Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR- radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild-type azurin. In the His46Gly and His117Gly mutants, a water molecule is co-ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild-type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin-imidazole complex is formed and the intramolecular electron-transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron-transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild-type and single-site-mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron-transfer pathways.

Intramolecular electron transfer (ET) between the CuA center and heme a in bovine cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methyl nicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 830 nm. After the initial reduction phase, the 830 nm absorption was partially restored, corresponding to reoxidation of the CuA center. Concomitantly, the absorption at 445 nm and 605 nm increased, indicating reduction of heme a. The rate constants for heme a reduction and CuA reoxidation were identical within experimental error and independent of the enzyme concentration. This demonstrates that a fast intramolecular electron equilibration is taking place between CuA and heme a. The rate constants for CuA --> heme a ET and the reverse (heme a --> CuA) process were found to be 13 000 s-1 and 3700 s-1, respectively, at 25 degrees C and pH 7.4. This corresponds to an equilibrium constant of 3.4 under these conditions. Thermodynamic and activation parameters of the ET reactions were determined. The significance of these results, particularly the observed low activation barriers, are discussed within the framework of the known three-dimensional structure, ET pathways and reorganization energies.

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.

The recent expression of an azurin mutant where the blue type 1 copper site is replaced by the purple CuA site of Paracoccus denitrificans cytochrome c oxidase has yielded an optimal system for examining the unique electron mediation properties of the binuclear CuA center, because both type 1 and CuA centers are placed in the same location in the protein while all other structural elements remain the same. Long-range electron transfer is induced between the disulfide radical anion, produced pulse radiolytically, and the oxidized binuclear CuA center in the purple azurin mutant. The rate constant of this intramolecular process, kET = 650 +/- 60 s-1 at 298 K and pH 5.1, is almost 3-fold faster than for the same process in the wild-type single blue copper azurin from Pseudomonas aeruginosa (250 +/- 20 s-1), in spite of a smaller driving force (0.69 eV for purple CuA azurin vs. 0.76 eV for blue copper azurin). The reorganization energy of the CuA center is calculated to be 0.4 eV, which is only 50% of that found for the wild-type azurin. These results represent a direct comparison of electron transfer properties of the blue and purple CuA sites in the same protein framework and provide support for the notion that the binuclear purple CuA center is a more efficient electron transfer agent than the blue single copper center because reactivity of the former involves a lower reorganization energy.

Pulse radiolytic reduction of disulfide bridges in ceruloplasmin yielding RSSR(-) radicals induces a cascade of intramolecular electron transfer (ET) processes. Based on the three-dimensional structure of ceruloplasmin identification of individual kinetically active disulfide groups and type 1 (T1) copper centers, the following is proposed. The first T1 copper(II) ion to be reduced in ceruloplasmin is the blue copper center of domain 6 (T1A) by ET from RSSR(-) of domain 5. The rate constant is 28 +/- 2 s(-1) at 279 K and pH 7.0. T1A is in close covalent contact with the type 3 copper pair and indeed electron equilibration between T1A and the trinuclear copper center in the domain 1-6 interface takes place with a rate constant of 2.9 +/- 0.6 s(-1). The equilibrium constant is 0.17. Following reduction of T1A Cu(II), another ET process takes place between RSSR(-) and T1B copper(II) of domain 4 with a rate constant of 3.9 +/- 0.8. No reoxidation of T1B Cu(I) could be resolved. It appears that the third T1 center (T1C of domain 2) is not participating in intramolecular ET, as it seems to be in a reduced state in the resting enzyme.

Azide binding to the blue copper oxidases laccase and ascorbate oxidase (AO) was investigated by electron paramagnetic resonance (EPR) and pulsed electron-nuclear double resonance (ENDOR) spectroscopies. As the laccase : azide molar ratio decreases from 1:1 to 1:7, the intensity of the type 2 (T2) Cu(II) EPR signal decreases and a signal at g approximately 1.9 appears. Temperature and microwave power dependent EPR measurements showed that this signal has a relatively short relaxation time and is therefore observed only below 40 K. A g approximately 1.97 signal, with similar saturation characteristics was found in the AO : azide (1:7) sample. The g < 2 signals in both proteins are assigned to an S = 1 dipolar coupled Cu(II) pair whereby the azide binding disrupts the anti-ferromagnetic coupling of the type 3 (T3) Cu(II) pair. Analysis of the position of the g < 2 signals suggests that the distance between the dipolar coupled Cu(II) pair is shorter in laccase than in AO. The proximity of T2 Cu(II) to the S = 1 Cu(II) pair enhances its relaxation rate, reducing its signal intensity relative to that of native protein. The disruption of the T3 anti-ferromagnetic coupling occurs only in part of the protein molecules, and in the remaining part a different azide binding mode is observed. The 130 K EPR spectra of AO and laccase with azide (1:7) exhibit, in addition to an unperturbed T2 Cu(II) signal, new features in the g parallel region that are attributed to a perturbed T2 in protein molecules where the anti-ferromagnetic coupling of T3 has not been disrupted. While these features are also apparent in the AO : azide sample at 10 K, they are absent in the EPR spectra of the laccase : azide sample measured in the range of 6-90 K. Moreover, pulsed ENDOR measurements carried out at 4.2 K on the latter exhibited only a reduction in the intensity of the 20 MHz peak of the 14N histidine coordinated to the T2 Cu(II) but did not resolve any significant changes that could i

The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis. The reversible ET reaction proceeds with a rate constant kET = k(1-->2) + k(2-->1) of 450 +/- 30 s(-1) at pH 7.0 and 298 K. The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k(1-->2) = 185 +/- 12 s(-1) and k(2-->1) 265 +/- 18 s(-1). The temperature dependence of K allowed us to determine the deltaH(o) value of the ET equilibrium to be 12.1 kJ mol(-1). Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, deltaH* = 22.7 +/- 3.4 kJ mol(-1) and deltaS* = -126 +/- 11 J K(-1) mol(-1); backward reaction, deltaH* = 10.6 +/- 1.7 kJ mol(-1) and deltaS* = -164 +/- 15 J K(-1) mol(-1). X-ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys136, which provides the thiolate ligand to the type 1 copper ion, and the adjacent His135 residue with its imidazole being one of the ligands to the type 2 Cu ion. This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase.

Intramolecular electron transfer from the type 1 copper center to the type 3 copper(II) pair is induced in the multi-copper enzyme, ascorbate oxidase, following pulse radiolytic reduction of the type 1 Cu(II) ion. In the presence of a slight excess of dioxygen over ascorbate oxidase, interaction between the trinuclear copper center and O2 is observed even with singly reduced ascorbate oxidase molecules. Under these conditions, the rate constant for intramolecular electron transfer from type 1 Cu(I) to type 3 Cu(II) increases 5-fold to 1100 +/- 300 s-1 (20 degrees C, pH 5.8) as compared to that of the same process under anaerobic conditions. This observation constitutes evidence for control of the internal electron transfer process by one of its substrates. The structural and functional significance of these findings are discussed.

Intramolecular long-range electron transfer (ET) processes have been investigated in two types of blue copper proteins; the single-copper protein, azurin and the multi-copper oxidase, ascorbate oxidase. These have several advantages for investigating the parameters that control the above reactions: (1) Their sole physiological role is mediating or catalyzing ET processes via the evolutionary optimized copper sites. (2) The three-dimensional structures of a considerable number of blue single copper containing proteins, e.g. azurins, and of ascorbate oxidase, have been determined at high resolution. (3) These proteins have no other cofactors except for the copper ions, thus the role of the polypeptide matrix can be addressed in a more straightforward manner. In azurins, the ET from the cystine (3-26) radical-ion produced by pulse-radiolytic reduction of this single disulfide bridge, to the Cu(II) ion bound at a distance of approximately 2.6 nm has been studied, in naturally occurring and in single-site mutated azurins. The role of changing specific amino acid residues on the internal long-range electron transfer (LRET) process and its potential pathways has been investigated. It is noteworthy that this process is most probably not part of the physiological function of azurin, hence, there has not been any evolutionary selection of structural elements for the reaction. Therefore, this provides a system for an unbiased examination of structural and chemical requirements for control of this process. By contrast, in blue copper oxidases, the internal ET from the electron uptake site from substrate to the O2 reduction site is part of these enzymes catalytic cycle, presumably optimized by selective pressure. We are investigating this internal ET in ascorbate oxidase and try to resolve the relation between this enzyme's distinct functional states and the internal ET rates. We conclude that in both types of proteins, the investigated LRET proceed primarily along covalent p

Single-site mutants of the blue, single-copper protein, azurin, from Pseudomonas aeruginosa were reduced by CO2- radicals in pulse radiolysis experiments. The single disulfide group was reduced directly by CO2- with rates similar to those of the native protein [Farver, O., & Pecht, I. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6968-6972]. The RSSR- radical produced in the above reaction was reoxidized in a slower intramolecular electron-transfer process (30-70 s-1 at 298 K) concomitant with a further reduction of the Cu(II) ion. The temperature dependence of the latter rates was determined and used to derive information on the possible effects of the mutations. The substitution of residue Phe114, situated on the opposite side of Cu relative to the disulfide, by Ala resulted in a rate increase by a factor of almost 2. By assuming that this effect is only due to an increase in driving force, lambda = 135 kJ mol-1 for the reorganization energy was derived. When Trp48, situated midway between the donor and the acceptor, was replaced by Leu or Met, only a small change in the rate of intramolecular electron transfer was observed, indicating that the aromatic residue in this position is apparently only marginally involved in electron transfer in wild-type azurin. Pathway calculations also suggest that a longer, through-backbone path is more efficient than the shorter one involving Trp48. The former pathway yields an exponential decay factor, beta, of 6.6 nm-1. Another mutation, raising the electron-transfer driving force, was produced by changing the Cu ligand Met121 to Leu, which increases the reduction potential by 100 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azu

Anaerobic reduction kinetics of the zucchini squash ascorbate oxidase (AO; L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) by pulse radiolytically produced CO2- radical ions were investigated. Changes in the absorption bands of type 1 [Cu(II)] (610 nm) and type 3 [Cu(II)] (330 nm) were monitored over a range of reactant concentrations, pH, and temperature. The direct bimolecular reduction of type 1 [Cu(II)] [(1.2 +/- 0.2) x 10(9) M-1.s-1] was followed by its subsequent reoxidation in three distinct phases, all found to be unimolecular processes with the respective specific rates of 201 +/- 8, 20 +/- 4, and 2.3 +/- 0.2 s-1 at pH 5.5 and 298 K. While at this pH no direct bimolecular reduction was resolved in the 330-nm band, at pH 7.0 such a direct process was observed [(6.5 +/- 1.2) x 10(8) M-1.s-1]. In the same slower time domains where type 1 [Cu(I)] reoxidation was monitored, reduction of type 3 [Cu(II)] was observed, which was also concentration independent and with identical rate constants and amplitudes commensurate with those of type 1 [Cu(II)] reoxidation. These results show that after electron uptake by type 1 [Cu(II)], its reoxidation takes place by intramolecular electron transfer to type 3 [Cu(II)]. The observed specific rates are similar to values reported for the limiting-rate constants of AO reduction by excess substrate, suggesting that internal electron transfer is the rate-determining step of AO activity. The temperature dependence of the intramolecular electron transfer rate constants was measured from 275 to 308 K at pH 5.5 and, from the Eyring plots, low activation enthalpies were calculated--namely, 9.1 +/- 1.1 and 6.8 +/- 1.0 kJ.mol-1 for the fastest and slowest phases, respectively. The activation entropies observed for these respective phases were -170 +/- 9 and -215 +/- 16 J.K-1.mol-1. The exceptionally low enthalpy barriers imply the involvement of highly optimized electron transfer pathways for internal electron transfer.

Kemi

Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkable specificity. The electron transfer is attained through weak electronic interaction between the active sites, so that considerable research efforts are centered on resolving the factors that control the rates of long-distance electron transfer reactions in proteins. These factors include (in addition to the distance and nature of the microenvironment separating the reactants) thermodynamic driving force and the configurational changes required upon reaction. Several of these aspects are addressed in this review, which is based primarily on recent work performed by the authors on model systems of blue copper-containing proteins. These proteins serve almost exclusively in electron transfer reactions, and as it turns out, their metal coordination sites are endowed with properties uniquely optimized for their function.