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The functioning of cytochrome c oxidases involves orchestration of long-range electron transfer (ET) events among the four redox active metal centers. We report the temperature dependence of electron transfer from the Cu A r site to the low-spin heme-( a ) b o site, i.e., Cu A r + heme- a ( b ) o → Cu A o + heme- a ( b ) r in three structurally characterized enzymes: A-type aa 3 from Paracoccus denitrificans (PDB code 3HB3) and bovine heart tissue (PDB code 2ZXW), and the B-type ba 3 from T. thermophilus (PDB codes 1EHK and 1XME). k , T data sets were obtained with the use of pulse radiolysis as described previously. Semiclassical Marcus theory revealed that λ varies from 0.74 to 1.1 eV, H ab , varies from ∼2 × 10 -5 eV (0.16 cm -1 ) to ∼24 × 10 -5 eV (1.9 cm -1 ), and β D varies from 9.3 to 13.9. These parameters are consistent with diabatic electron tunneling. The II-Asp111Asn Cu A mutation in cytochrome ba 3 had no effect on the rate of this reaction whereas the II-Met160Leu Cu A -mutation was slower by an amount corresponding to a decreased driving force of ∼0.06 eV. The structures support the presence of a common, electron-conducting "wire" between Cu A and heme- a ( b ). The transfer of an electron from the low-spin heme to the high-spin heme, i.e., heme- a ( b ) r + heme- a 3 o → heme- a ( b ) o + heme- a 3 r , was not observed with the A-type enzymes in our experiments but was observed with the Thermus ba 3 ; its Marcus parameters are λ = 1.5 eV, H ab = 26.6 × 10 -5 eV (2.14 cm -1 ), and β D = 9.35, consistent also with diabatic electron tunneling between the two hemes. The II-Glu15Ala mutation of the K-channel structure, ∼ 24 Å between its CA and Fe- a 3 , was found to completely block heme- b r to heme- a 3 o electron transfer. A structural mechanism is suggested to explain these observations.

The Marcus theory of electron transfer (ET) predicts that while the ET rate constants increase with rising driving force until it equals a reaction's reorganization energy, at higher driving force the ET rate decreases, having reached the Marcus inverted region. While experimental evidence of the inverted region has been reported for organic and inorganic ET reactions as well as for proteins conjugated with ancillary redox moieties, evidence of the inverted region in a "protein-only" system has remained elusive. We herein provide such evidence in a series of nonderivatized proteins. These results may facilitate the design of ET centers for future applications such as advanced energy conversions.

The multicopper oxidases are an intriguing, widespread family of enzymes that catalyze the reduction of O2 to water by a variety of single-electron and multiple-electron reducing agents. The structure and properties of the copper binding sites responsible for the latter chemical transformations have been studied for over 40 years and a detailed picture is emerging. This review focuses particularly on the kinetics of internal electron transfer between the type 1 (blue) copper site and the trinuclear center, as well as on the nature of the intermediates formed in the oxygen reduction process.

Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.

Rate constants and activation parameters have been determined for the internal electron transfer from type 1 (T1) to type 3 (T3) copper ions in laccase from both the fungus Trametes hirsuta and the lacquer tree Rhus vernicifera, using the pulse radiolysis method. The rate constant at 298 K and the enthalpy and entropy of activation were 25 ± 1 s(-1), 39.7 ± 5.0 kJ·mol(-1) and -87 ± 9 J·mol(-1) ·K(-1) for the fungal enzyme and 1.1 ± 0.1 s(-1), 9.8 ± 0.2 kJ·mol(-1) and -211 ± 3 J·mol(-1) ·K(-1) for the tree enzyme. The initial reduction of the T1 site by pulse radiolytically produced radicals was direct in the case of T. hirsuta laccase, but occured indirectly via a disulfide radical in R. vernicifera. The equilibrium constant that characterizes the electron transfer from T1 to T3 copper ions was 0.4 for T. hirsuta laccase and 1.5 for R. vernicifera laccase, leading to full reduction of the T1 site occurring at 2.9 ± 0.2 electron equivalents for T. hirsuta and 4 electron equivalents for R. vernicifera laccase. These results were compared with each other and with those for the same process in other multicopper oxidases, ascorbate oxidase and Streptomyces coelicolor laccase, using available structural information and electron transfer theory., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Type zero copper is a hard-ligand analogue of the classical type 1 or blue site in copper proteins that function as electron transfer (ET) agents in photosynthesis and other biological processes. The EPR spectroscopic features of type zero Cu(II) are very similar to those of blue copper, although lacking the deep blue color, due to the absence of thiolate ligation. We have measured the rates of intramolecular ET from the pulse radiolytically generated C3-C26 disulfide radical anion to the Cu(II) in both type zero C112D/M121L and type 2 C112D Pseudomonas aeruginosa azurins in pH 7.0 aqueous solutions between 8 and 45 °C. We also have obtained rate/temperature (10-30 °C) profiles for ET reactions between these mutants and the wild-type azurin. Analysis of the rates and activation parameters for both intramolecular and intermolecular ET reactions indicates that the type zero copper reorganization energy falls in a range (0.9-1.1 eV) slightly above that for type 1 (0.7-0.8 eV), but substantially smaller than that for type 2 (>2 eV), consistent with XAS and EXAFS data that reveal minimal type zero site reorientation during redox cycling., (© 2011 American Chemical Society)

Control of electron transfer rates, caused by intrinsic protein structural properties, is an intriguing feature of internal biological electron transfer (ET) reactions. The small laccase (SLAC) isolated from Streptomyces coelicolor has recently been shown to have structural and reactivity features distinct from those of other laccases. While other copper oxidases contain three cupredoxin domains, the SLAC 3D structure has recently been determined and shown to consist of only two, and a different reaction intermediate has been reported for it. It was therefore of particular interest to investigate the intramolecular ET between the type 1 and the trinuclear copper center in SLAC which is a crucial step in the catalytic cycle of the multicopper oxidases, leading to dioxygen reduction to water. This ET step was found to markedly depend on the reduction state of the enzyme, possibly reflecting site-site interactions so far not observed in other multicopper oxidases.

The cd(1) nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme-c and one heme-d(1). Heme-c is the electron entry site, whereas heme-d(1) constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d(1) hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme-c was initially reduced, in an essentially diffusion-controlled bimolecular process, followed by unimolecular electron equilibration between the c and d(1) hemes (k(ET) = 4.3 s(-1) and K = 1.4 at 298 K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by approximately 30-fold) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd(1) nitrite reductases, and we discuss this control based on the available 3D structure of the wild-type and the H369A mutant, in the reduced and oxidized states.

The redox reactivity of the three disulfide bridges and the flavin present in each protomer of the wild-type Arabidopsis thaliana mitochondrial sulfhydryl oxidase (AtErv1) homodimer has been investigated. Pulse radiolytically produced CO2- radical ions were found to reduce the disulfide bridges to yield disulfide radicals, RSS*R-. Rates and absorption changes due to formation or decay of RSS*R- and the flavin quinone, semiquinone, and hydroquinone were measured and analyzed. During the first 100 micros following the pulse, the flavin was reduced to the semiquinone by intramolecular electron transfer from the active site disulfide radical. The semiquinone and the remaining disulfide radicals then reacted by much slower, 40 ms to 40 s, inter-homodimer electron transfer reactions, culminating in reduced flavin and dithiols. The dithiols were then subject to oxidation by enzyme molecules via their intrinsic enzymatic activity, at a rate comparable to the slower intermolecular processes in the 10-s time regime. Mutants of AtErv1 lacking each of the three individual cysteine pairs were studied to determine the involvement of the respective disulfide groups in these reactions. Elimination of the active site disulfide bridge increased the stability of the flavin semiquinone making it a long-lived product. Relevance of these observations to the design and function of the sulfhydryl oxidases is discussed.

Mixed valence (MV) coordination compounds play important roles in redox reactions in chemistry and biology. Details of the contribution of a mixed valence state to protein electron transfer (ET) reactivity such as reorganization energy, however, have not been experimentally defined. Herein we report measurements of reorganization energies of a binuclear CuA center engineered into Pseudomonas aeruginosa azurin that exhibits a reversible transition between a totally delocalized MV state at pH 8.0 and a trapped valence (TV) state at pH 4.0. The reorganization energy of a His120Ala variant of CuA azurin that displays a TV state at both the above pH values has also been determined. We found that the MV-to-TV state transition increases the reorganization energy by 0.18 eV, providing evidence that the MV state of the CuA center has lower reorganization energy than its TV counterpart. We have also shown that lowering the pH from 8.0 to 4.0 results in a similar (approximately 0.4 eV) decrease in reorganization energy for both blue (type 1) and purple (CuA) azurins, even though the reorganization energies of the two different copper centers are different at a given pH. These results suggest that the MV state plays only a secondary role in modulation of the ET reactivity via the reorganization energy, as compared to that of the driving force.

Copper-containing nitrite reductase harbors a type-1 and a type-2 Cu site. The former acts as the electron acceptor site of the enzyme, and the latter is the site of catalytic action. The effect of the methionine ligand on the reorganization energy of the type-1 site was explored by studying the electron-transfer kinetics between NiR (wild type (wt) and the variants Met150Gly and Met150Thr) with Fe(II)EDTA and Fe(II)HEDTA. The mutations increased the reorganization energy by 0.3 eV (30 kJ mol-1). A similar increase was found from pulse radiolysis experiments on the wt NIR and three variants (Met150Gly, Met150His, and Met150Thr). Binding of the nearby Met62 to the type-1 Cu site in Met150Gly (under influence of an allosteric effector) lowered the reorganization energy back to approximately the wt value. According to XRD data the structure of the reduced type-1 site in Met150Gly NiR in the presence of an allosteric effector is similar to that in the reduced wt NiR (solved to 1.85 A), compatible with the similarity in reorganization energy.

The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent.

Intramolecular electron transfer between CuA and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. CuA, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the CuA center and the concomitant 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 and its degree of reduction, demonstrating 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 20,400 s(-1) and 10,030 s(-1), respectively, at 25 degrees C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme's known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.

A biomimetic long-range electron transfer (ET) system consisting of the blue copper protein azurin, a tunneling barrier bridge, and a gold single-crystal electrode was designed on the basis of molecular wiring self-assembly principles. This system is sufficiently stable and sensitive in a quasi-biological environment, suitable for detailed observations of long-range protein interfacial ET at the nanoscale and single-molecule levels. Because azurin is located at clearly identifiable fixed sites in well controlled orientation, the ET configuration parallels biological ET. The ET is nonadiabatic, and the rate constants display tunneling features with distance-decay factors of 0.83 and 0.91 A(-1) in H(2)O and D(2)O, respectively. Redox-gated tunneling resonance is observed in situ at the single-molecule level by using electrochemical scanning tunneling microscopy, exhibiting an asymmetric dependence on the redox potential. Maximum resonance appears around the equilibrium redox potential of azurin with an on/off current ratio of approximately 9. Simulation analyses, based on a two-step interfacial ET model for the scanning tunneling microscopy redox process, were performed and provide quantitative information for rational understanding of the ET mechanism.

The copper- and heme-containing nitrite reductases (NiRs) are key enzymes in denitrification. Their subunits contain two distinct redox-active metal centers, an electron-accepting site and a nitrite-reducing site, to carry out the single-electron reduction of nitrite to nitric oxide. Catalytic cycles of both enzyme families employ intramolecular electron transfer that can be rate-determining for their activity. Herein, we report results comparing these two enzyme families in order to resolve the different mechanisms controlling intramolecular electron transfer in these proteins.

Pulse radiolysis has been employed to investigate the intramolecular electron transfer (ET) between the type 1 (T1) and type 2 (T2) copper sites in the Met144Ala Alcaligenes xylosoxidans nitrite reductase (AxCuNiR) mutant. This mutation increases the reduction potential of the T1 copper center. Kinetic results suggest that the change in driving force has a dramatic influence on the reactivity: The T2Cu(II) is initially reduced followed by ET to T1Cu(II). The activation parameters have been determined and are compared with those of the wild-type (WT) AxCuNiR. The reorganization energy of the T2 site in the latter enzyme was calculated to be 1.6+/-0.2 eV which is two-fold larger than that of the T1 copper center in the WT protein.

Cytochrome cd1 nitrite reductase is a bifunctional multiheme enzyme catalyzing the one-electron reduction of nitrite to nitric oxide and the four-electron reduction of dioxygen to water. Kinetics and thermodynamics of the internal electron transfer process in the Pseudomonas stutzeri enzyme have been studied and found to be dominated by pronounced interactions between the c and the d1 hemes. The interactions are expressed both in dramatic changes in the internal electron-transfer rates between these sites and in marked cooperativity in their electron affinity. The results constitute a prime example of intraprotein control of the electron-transfer rates by allosteric interactions.

High field (W-band, 95 GHz) pulsed electron-nuclear double resonance (ENDOR) measurements were carried out on a number of proteins that contain the mixed-valence, binuclear electron-mediating Cu(A) center. These include nitrous oxide reductase (N(2)OR), the recombinant water-soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase (COX) ba(3) (M160T9), its M160QT0 mutant, where the weak axial methionine ligand has been replaced by a glutamine, and the engineered "purple" azurin (purpAz). The three-dimensional (3-D) structures of these proteins, apart from the mutant, are known. The EPR spectra of all samples showed the presence of a mononuclear Cu(II) impurity with EPR characteristics of a type II copper. At W-band, the g( perpendicular) features of this center and of Cu(A) are well resolved, thus allowing us to obtain a clean Cu(A) ENDOR spectrum. The latter consists of two types of ENDOR signals. The first includes the signals of the four strongly coupled cysteine beta-protons, with isotropic hyperfine couplings, A(iso), in the 7-15 MHz range. The second group consists of weakly coupled protons with a primarily anisotropic character with A(zz) < 3 MHz. Orientation selective ENDOR spectra were collected for N(2)OR, M160QT0, and purpAz, and simulations of the cysteine beta-protons signals provided their isotropic and anisotropic hyperfine interactions. A linear correlation with a negative slope was found between the maximum A(iso) value of the beta-protons and the copper hyperfine interaction. Comparison of the best-fit anisotropic hyperfine parameters with those calculated from dipolar interactions extracted from the available 3-D structures sets limit to the sulfur spin densities. Similarly, the small coupling spectral region was simulated on the basis of the 3-D structures and compared with the experimental spectra. It was found that the width of the powder patterns of the weakly coupled protons recorded at g(perpendicular) is mainly determined by the histidine H(epsilon)(1) protons. Furthermore, the splitting in the outer wings of these powder patterns indicates differences in the positions of the imidazole rings relative to the Cu(2)S(2) core. Comparison of the spectral features of the weakly coupled protons of M160QT0 with those of the other investigated proteins shows that they are very similar to those of purpAz, where the Cu(A) center is the most symmetric, but the copper spin density and the H(epsilon)(1)-Cu distances are somewhat smaller. All proteins show the presence of a proton with a significantly negative A(iso) value which is assigned to an amide proton of one of the cysteines. The simulations of both strongly and weakly coupled protons, along with the known copper hyperfine couplings, were used to estimate and compare the spin density distribution in the various Cu(A) centers. The largest sulfur spin density was found in M160T9, and the lowest was found in purpAz. In addition, using the relation between the A(iso) values of the four cysteine beta-protons and the H-C-S-S dihedral angles, the relative contribution of the hyperconjugation mechanism to A(iso) was determined. The largest contribution was found for M160T9, and the lowest was found for purpAz. Possible correlations between the spin density distribution, structural features, and electron-transfer functionality are finally suggested.

Cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri catalyzes the one electron reduction of nitrite to nitric oxide. It is a homodimer, each monomer containing one heme-c and one heme-d(1), the former being the electron uptake site while the latter is the nitrite reduction site. Hence, internal electron transfer between these sites is an inherent element in the catalytic cycle of this enzyme. We have investigated the internal electron transfer reaction employing pulse radiolytically produced N-methyl nicotinamide radicals as reductant which reacts solely with the heme-c in an essentially diffusion controlled process. Following this initial step, the reduction equivalent is equilibrating between the c and d(1) heme sites in a unimolecular process (k=23 s(-1), 298 K, pH 7.0) and an equilibrium constant of 1.0. The temperature dependence of this internal electron transfer process has been determined over a 277-313 K temperature range and yielded both equilibrium standard enthalpy and entropy changes as well as activation parameters of the specific rate constants. The significance of these parameters obtained at low degree of reduction of the enzyme is discussed and compared with earlier studies on cd(1) nitrite reductases from other sources.

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.

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 indicate azide binding to this ion. The lack of T2 Cu(II) signal perturbation below 90 K in laccase may be due to temperature dependence of the coupling within the trinuclear : azide complex.

Cu2+, Ni2+, Zn2+, Co2+ and Cd2+ were evaluated in metal ion affinity chromatography for enrichment of selenoprotein P, and immobilized Co2+ affinity chromatography was found to be the most selective chromatographic method. The chromatography was performed by fast protein liquid chromatography and the fractionation was followed by analysis of the collected fractions for selenium by inductively coupled plasma mass spectrometry. By the combination of immobilized Co2+ affinity chromatography and heparin affinity chromatography a simple method was developed yielding a 14,800-fold enrichment of selenoprotein P. The purity of the protein was determined by SDS-PAGE and by sequencing from polyvinylidene difluoride blots of SDS-PAGE gels.

We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.

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.

The cDNA, coding for the first metal-binding domain (MBD1) of Menkes protein, was cloned into the T7-system based vector, pCA. The T7 lysozyme-encoding plasmid, pLysS, is shown to be crucial for expression, suggesting that the protein is toxic to the cells. Adding copper to the growth medium did not affect the plasmid stability. MBD1 is purified in two steps with a typical yield of 12 mg.L-1. Menkes protein, a P-type ATPase, contains a sequence GMXCXSC that is repeated six times, at the N-terminus. The paired cysteine residues are involved in metal binding. MBD1 has only two cysteine residues, which can exist as free thiol groups (reduced), as a disulphide bond (oxidized) or bound to a metal ion [e.g. Cu(I)-MBD1]. These three MBD1 forms have been investigated using CD. No major spectral change was seen between the different MBD1 forms, indicating that the folding is not changed upon metal binding. A copper-bound MBD1 was also studied by EPR, and the lack of an EPR signal suggests that the oxidation state of copper bound to MBD1 is Cu(I). Cu(I) binding studies were performed by equilibrium dialysis and revealed a stoichiometry of 1 : 1 and an apparent Kd = 46 microM. Oxidized MBD1, however, is not able to bind copper. Different copper complexes were investigated for their ability to reconstitute apo-MBD1. Given the same total copper concentration CuCl43- was superior to Cu(I)-thiourea (structural analogue of metallothionein) and Cu(I)-glutathione (used at fivefold higher copper concentration) although the latter two were able to partially reconstitute apo-MBD1. Cu(II) was not able to reconstitute apo-MBD1, presumably due to Cu(II)-induced oxidation of the thiol groups. Based on our results, glutathione and/or metallothionein are likely candidates for the in vivo incorporation of copper to Menkes protein.

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.

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.