Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c. Optical multichannel detection

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex was investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-1-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. Time-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms between 325 and 650 nm. On the basis of singular value decomposition (SVD) and multiexponential fitting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of approximately 2.0 x 10(4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed by faster electron transfer from cytochrome c to Cu(A), with the equilibrium favoring the reduced cytochrome c. Subsequently, Cu(A) equilibrates with heme a with an apparent rate constant of approximately 1 x 10(4) s(-1). On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the postulated intermediates, supporting the proposed mechanism.     

Photophysics of metalloazurins

The fluorescence lifetimes of Cu(II), Cu(I), Ag(I), Hg(II), Co(II), and Ni(II) azurin Pae from Pseudomonas aeruginosa and Cu(II), Cu(I), and Hg(II) azurin Afe from Alcaligenes faecalis were measured at 295 K by time-correlated single-photon counting. In addition, fluorescence lifetimes of Cu(II) azurin Pae were measured between 30 and 160 K and showed little change in value. Ultraviolet absorption difference spectra between metalloazurin Pae and apoazurin Pae were measured, as were the fluorescence spectra of metalloazurins. These spectra were used to determine the spectral overlap integral required for dipole-dipole resonance calculations. All metalloazurins exhibit a reduced fluorescence lifetime compared to their respective apoazurins. Forster electronic energy transfer rates were calculated for both metalloazurin Pae and metalloazurin Afe derivatives; both enzymes contain a single tryptophyl residue which is located in a different position in the two azurins. These azurins have markedly different fluorescence spectra, and electronic energy transfers occur from these two tryptophyl sites with different distances and orientations and spectral overlap integral values. Intramolecular distances and orientations were derived from an X-ray crystallographic structure and a molecular dynamic simulation of the homologous azurin Ade from Alcaligenes denitrificans, which contains both tryptophyl sites. Assignments were made of metal-ligand-field electronic transitions and of transition dipole moments and directions for tryptophyl residues, which accounted for the observed fluorescence quenching of Hg(II), Co(II), and Ni(II) azurin Pae and Cu(II) and Hg(II) azurin Afe. The fluorescence of azurin Pae is assigned as a 1Lb electronic transition, while that of azurin Afe is 1La. The marked fluorescence quenching of Cu(II) azurin Pae and Cu(I) azurin Pae and Afe is less well reproduced by our calculations, and long-range oxidative and reductive electron transfer, respectively, are proposed as additional quenching mechanisms. This study illustrates the application of Forster electronic energy transfer calculations to intramolecular transfers in structurally well characterized molecular systems and demonstrates its ability to predict observed fluorescence quenching rates when the necessary extensive structural, electronic transition assignment, and spectroscopic data are available. The agreement between Forster calculations and quenching rates derived from fluorescence lifetime measurements suggests there are limited changes in conformation between crystal structure and solution structures, with the exception of the tryptophyl residue of azurin Afe, where a conformation derived from a molecular simulation in water was necessary rather than that found in the crystal structure.     

Resolution of two distinct electron transfer sites on azurin

Pseudomonas aeruginosa azurin is stoichiometrically and specifically labeled upon reduction by Cr(II)aq ions, yielding a substitution-inert Cr(III) adduct on the protein surface. We investigated the effect of this chemical modification on the reactivity of azurin with two of its presumed partners in the redox system of the bacterium. The Pseudomonas cytochrome oxidase catalyzed oxidation of reduced native and Cr(III)-labeled azurin by O2 was found to be unaffected by the modification. The kinetics of the electron exchange reaction between native or Cr(III)-labeled azurin and cytochrome c551 were studied by the temperature-jump method. Though similar chemical relaxation spectra were observed for native and modified systems, they differ quantitatively. Analysis of the concentration dependences of the relaxation times and amplitudes showed that both obey the same mechanism but that the specific reaction rates of the Cr(III)-modified protein are attenuated. This decreased reactivity of Cr(III)-labeled azurin toward one of its physiological partners suggests the involvement of the labeled region in the electron transfer reaction with cytochrome c551. Furthermore, the presence of a second active site, involved in the reduction of cytochrome oxidase, is suggested by the results.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor Q(A)

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron--quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D|=0.0286 cm(-1), |E|=0.0044 cm(-1) was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T=100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron--quinone primary acceptor complex.     

Spectroscopic evidence for interactions between hexacyanoiron(II/III) and an engineered purple CuA azurin

Interactions between hexacyanoiron(II/III) and a dinuclear, mixed valence Cu(A) center in engineered Cu(A) azurin have been investigated by UV-visible (UV-vis) and electron paramagnetic resonance (EPR) spectroscopic techniques. Addition of ferricyanide (hexacyanoiron(III)) to the Cu(A) azurin resulted in a new absorption band around 500 nm in the UV-vis and an isotropic line at g = 2.16 in the EPR spectra. Control experiments, including additions of Cu(II)SO(4) or Cu(I)(CH(3)CN)(4)PF(6) to ferricyanide or ferrocyanide, as well as gel filtration purification of the ferricyanide-Cu(A) azurin adduct indicate complex formation between cupric ion and ferrocyanide ion in the protein. Solvent or small molecule accessibility, metal oxidation state and the presence of more than one metal ion are potential factors important for the complex formation. These findings must be taken into consideration when using ferricyanide or ferrocyanide as redox agents for studying Cu(A) centers in proteins.     

Metal-binding properties of an engineered purple CuA center in azurin

A CUA center engineered into Pseudomonas aeruginosa azurin was studied by metal substitution. Metal-binding properties were determined by electronic absorption (UV-vis) and electrospray ionization mass spectrometry (ESI-MS). The metal-binding site readily binds thiophilic metal ions, such as Hg(II), Ag(I), Cu(I), Cd(II), and Au(I). Harder metal ions, like Co(II), bind to apo-CuA-azurin only under basic conditions (pH 9.1-9.2). The results obtained from these studies indicate that two factors influence metal binding in CuA azurin: (1) the site favors metal combinations which produce an overall +3 charge, and (2) the site binds soft, thiophilic metal ions. The results demonstrate the remarkable ability of the CuA center to maintain valence delocalization of its native metal ions and to ensure redox accessibility of only one of the two redox couples (i.e., [Cu(1.5)...Cu(1.5)]<==> [Cu(I)...Cu(I)]) under physiological conditions. These findings may lead to the preparation of new metal ion derivatives and can serve as a basis for understanding this efficient electron transfer center.     

Silver binding toPseudomonas aeruginosa azurin

Summary The interaction between azurin and silver ions was investigated, by means of ultraviolet, fluorescence and atomic absorption spectroscopies, as a function of the redox state of the protein. The Ag(I) ion has a very low affinity for oxidized azurin. Interestingly, the affinity is much higher for reduced azurin; in this case Ag(I) completely displaces the Cu(I) ion from the native binding site. The effect is very specific for silver ions since other ions, such as Hg(II), Ni(II) and Cd(II), do not produce the same effect. Treatment of reduced and oxidized azurin with excess Ag(I) (2-8-fold stoichiometric) shows that there is a second binding site for silver ions on the protein which can also bind Cu(II) and Hg(II) with comparable affinities.     

Photoinduced electron-transfer kinetics of singly labeled ruthenium bis(bipyridine) dicarboxybipyridine cytochrome c derivatives

Cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)] were prepared by using the procedure described previously [Pan, L. P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Four additional singly labeled derivatives were purified, bringing the total number to 10. These derivatives have a strong luminescence emission centered at 662 nm arising from the excited state, RuII*. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from RuII* to the ferric heme group (k1) and for the thermal back-reaction from the ferrous heme group to RuIII (k2). The rate constants were found to be k1 = 14 X 10(6) s-1 and k2 = 24 X 10(6) s-1 for the derivative modified at lysine 72, which has a distance of 8-16 A between the ruthenium and heme groups. Similar rate constants were found for the derivatives modified at lysines 13 and 27, which have distances of 6-12 A separating the ruthenium and heme groups. The rate constants were significantly slower for the derivatives modified at lysine 25 (k1 = 1 X 10(6) s-1, k2 = 1.5 X 10(6) s-1) and lysine 7 (k1 = 0.3 X 10(6) s-1, k2 = 0.5 X 10(6) s-1), which have distances of 9-16 A. Transients due to photoinduced electron transfer could not be detected for the remaining derivatives, which have larger distances between the ruthenium and heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoinduced electron transfer between cytochrome c peroxidase and horse cytochrome c labeled at specific lysines with (dicarboxybipyridine)(bisbipyridine)ruthenium(II)

The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) [Ru(II)] were studied by flash photolysis. All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI. This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI. The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively. The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives. These results suggest that there are two binding orientations for cytochrome c on CMPI. The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer. Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase. A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength. When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed. The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer.     

The structure of the Met144Leu mutant of copper nitrite reductase from <Emphasis Type="Italic">Alcaligenes xylosoxidans</Emphasis> provides the first glimpse of a protein–protein complex with azurin II

Cu-containing nitrite reductases (NiRs) perform the reduction of nitrite to NO via an ordered mechanism in which the delivery of a proton and an electron to the catalytic type 2 Cu site is highly orchestrated. Electron transfer from a redox partner protein, azurin or pseudoazurin, to the type 1 Cu site is assumed to occur through the formation of a protein-protein complex. We report here a new crystal form in space group P2(1)2(1)2(1) of the Met144Leu mutant of NiR from Alcaligenes xylosoxidans (AxNiR), revealing a head-to-head packing motif involving residues around the hydrophobic patch of domain 1. Superposition of the structure of azurin II with that of domain 1 of one of the Met144Leu molecules provides the first glimpse of an azurin II-NiR protein-protein complex. Mutations of two of the residues of AxNiR, Trp138His (Barrett et al. in Biochemistry 43:16311-16319, 2004) and Met87Leu, highlighted in the AxNiR-azurin complex, results in substantially decreased activity when azurin is used as the electron donor instead of methyl viologen, providing direct evidence for the importance of this region for complex formation.     

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols

Azurin from Pseudomona aeruginosa is a small copper protein with a single tryptophan (Trp) buried in the structure. The Gibbs free energies associated with the folding of holo azurin, calculated monitoring Trp fluorescence and changes in absorbance on the ligand-to-metal band, are different because these techniques probe their local environments, thereby being able to probe different conformational changes. The presence of an intermediate state was observed during the chemical denaturation of the protein. Upon denaturation, a 30-fold increase is observed in the magnitude of the quenching constant of the tryptophan fluorescence by acrylamide, because this residue becomes more accessible to the quencher. Entrapping the protein in sol-gel materials lowers its stability possibly because the solvation properties of the macromolecule are changed. The thermal denaturation of azurin immobilized in a sol-gel monolith is irreversible, which tends to rule out an aggregation mechanism to account for the irreversibility of the denaturation of the protein free in solution. Unlike the Cu(II) ion, the Gd(III) ion accommodates in site B of azurin with high affinity and the folding free energy of Gd-azurin is larger than that of apo azurin.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor QA

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron-quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D| = 0.0286 cm⁻¹, |E| = 0.0044 cm⁻¹ was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T = 100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron-quinone primary acceptor complex.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

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]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

Structure and reactivity of type-I copper sites

Summary Biological electron transfer is not well understood. The question is addressed in this contribution with reference to the so-called blue copper proteins, each of which has a single copper atom at its active centre. The redox activity (as probed by the electron self exchange reaction) of the Cu centre seems not to be affected. The electron self exchange reaction is known to proceed through His-117, and the hydrophobic patch is most important in the formation of the azurin/azurin encounter complex. Ph effects have not been observed on the three-dimensional structure ofA. denitrificans azurin, which may indicate that if present at all these have no direct physiological implications. Mutants are in process of construction.     

Preparation and characterization of singly labeled ruthenium polypyridine cytochrome c derivatives

A novel two-step procedure has been developed to prepare cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)]. In the first step, cytochrome c was treated with the mono-N-hydroxysuccinimide ester of 4,4'-dicarboxy-2,2'-bipyridine (dcbpy) to convert positively charged lysine amino groups to negatively charged dcbpy-lysine groups. Singly labeled dcbpy-cytochrome c derivatives were then separated and purified by ion-exchange chromatography. In the second step, the individual dcbpy-cytochrome c derivatives were treated with RuII(bpy)2CO3 to form singly labeled RuII(bpy)2(dcbpy-cytochrome c) derivatives. The specific lysine labeled in each derivative was determined by reverse-phase chromatography of a tryptic digest. All of the derivatives had a strong luminescence emission centered at 662 nm, but the luminescence decay rates were increased relative to that of a non-heme protein control, RuII(bpy)2(dcbpy-lysozyme), which was 1.8 X 10(6) s-1. The luminescence decay rates were found to be 21, 16, 7.2, 5.7, 4.3, 4.3, and 3.5 X 10(6) s-1 for derivatives singly labeled at lysines 13, 72, 25, 7, 39, 86, and 87, respectively. There was an inverse relationship between the luminescence decay rates and the distances between the ruthenium labels and the heme group. The increased luminescence decay rates observed in the cytochrome c derivatives might be due to electron transfer from the excited triplet state of ruthenium to the ferric heme group. However, it is also possible that an energy-transfer mechanism might contribute to the luminescence quenching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic effects on the kinetics of photoinduced electron-transfer reactions of the triplet state of zinc cytochrome c with wild-type and mutant forms of Pseudomonas aeruginosa azurin

We study, by laser flash photolysis, the effects of ionic strength on the kinetics of the reaction ³Zncyt + az(II) → Zncyt⁺ + az(I), i.e., oxidative quenching of the triplet state of zinc cytochrome c by the wild-type form and the following three mutants of cupriazurin: Met44Lys, Met64Glu, and the double mutant Met44Lys/Met64Glu. Mutations in the hydrophobic patch of azurin significantly affect the reactivity of the protein with the triplet state of zinc cytochrome c. Dependence on the ionic strength of the bimolecular rate constant for the aforementioned reaction is analyzed by several electrosatic models. The two transition-state theories, Brønsted-Debye-Hückel and van Leeuwen theories, allow the best approximation to the experimental data when effective charges of the proteins are used. Protein-protein interactions are also analyzed in terms of local charges on the protein surfaces. The rate constants depend little on ionic strength, and the monopolar and dipolar electrostatic interactions between zinc cytochrome c and azurin are not well resolved. Semiquantitative analysis of electrostatic interactions indicates that azurin uses its hydrophobic patch for contact with zinc cytochrome c.     

Copper Proteins and Oxygen : Correlations between structure and function of the copper oxidases

A comprehensive survey of the interaction of the copper proteins and oxygen is presented including a correlation of structure, function, and other properties of the known copper oxidases and of hemocyanin. The origin of their blue color and the structure of copper complexes and copper proteins are related to the oxidation state of copper ion and relevant electronic transitions probably arising from the formation of charge transfer complexes. The oxygen reactions of hemocyanin, ceruloplasmin, and cytochrome oxidase show half-saturation values far below the other Cu enzymes. The formation of hydrogen peroxide as a reaction product is associated with the presence of one Cu atom per oxidase molecule or catalytic system. Water is the corresponding product of the other Cu oxidases with four or more Cu atoms per molecule, except for monoamine oxidase. Mechanisms for the oxidase action of the two and four electron transfer Cu oxidases and tyrosinase are proposed. These reactions account for the number, the oxidation-reduction potential, and the oxidation state of Cu in the resting enzyme, the cyclical change from Cu(II) to Cu(I), the diatomic nature of O₂, the sequence of the oxidation and reduction reactions, and other salient features. The catalytic reactions involved in the oxidation of ascorbic acid by plant ascorbate oxidase, ceruloplasmin, and Cu(II) are compared. Finally the substrate specificity, inhibitory control, and the detailed mechanism of the oxidase activity of ceruloplasmin are summarized.     

Role of ligand substitution on long-range electron transfer in azurins.

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.