The metal site of Pseudomonas aeruginosa azurin, revealed by a crystal structure determination of the Co(II) derivative and Co-EPR spectroscopy.

The crystal structure of cobalt-substituted azurin from Pseudomonas aeruginosa has been determined to final crystallographic R value of 0.175 at 1.9 A resolution. There are four molecules in the asymmetric unit in the structure, and these four molecules are packed as a dimer of dimers. The dimer packing is very similar to that of the wild-type Pseudomonas aeruginosa azurin dimer. Replacement of the native copper by the cobalt ion has only small effects on the metal binding site presumably because of the existence of an extensive network of hydrogen bonds in its immediate neighborhood. Some differences are obvious, however. In wild-type azurin the copper atom occupies a distorted trigonal bipyramidal site, while cobalt similar to zinc and nickel occupy a distorted tetrahedral site, in which the distance to the Met121,S(delta) atom is increased to 3.3-3.5 A and the distance to the carbonyl oxygen of Gly45 has decreased to 2.1-2.4 A. The X-band EPR spectrum of the high-spin Co(II) in azurin is well resolved (apparent g values gx' = 5.23; gy' = 3.83; gz' = 1.995, and hyperfine splittings Ax' = 31; Ay' = 20-30; Az' = 53 G) and indicates that the ligand field is close to axial.

The type 2 copper of ascorbate oxidase.

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at -30 degrees C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500-3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at -30 degrees C the activity fell to 1400-2000 units in the phosphate buffer but only to 2600-2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at -30 degrees C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu2+ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu2+ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)6(3), large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.

Electron paramagnetic resonance studies of the soluble CuA protein from the cytochrome ba3 of Thermus thermophilus.

The electron paramagnetic resonance (EPR) spectrum of the binuclear CuA center in the water-soluble subunit II fragment from cytochrome ba3 of Thermus thermophilus was recorded at 3.93, 9.45, and 34.03 GHz, and the EPR parameters were determined by computer simulations. The frequency and M1 dependence of the linewidth was discussed in terms of g strain superimposed on a correlation between the A and g values. The g values were found to be gx = 1.996, gy = 2.011, gz = 2.187, and the two Cu ions contribute nearly equally to the hyperfine structure, with magnitude of Ax magnitude of approximately 15 G, magnitude of Ay magnitude = 29 G, and magnitude of Az magnitude of = 28.5 G (65Cu). Theoretical CNDO/S calculations, based on the x-ray structure of the Paracoccus denitrificans enzyme, yield a singly occupied antibonding orbital in which each Cu is pi*-bonded to one S and sigma*-bonded to the other. In contrast to the equal spin distribution suggested by the EPR simulations, the calculated contributions from the Cu ions differ by a factor of 2. However, only small changes in the ligand geometry are needed to reproduce the experimental results.

Mutant Met121Ala of Pseudomonas aeruginosa azurin and its azide derivative: crystal structures and spectral properties.

The crystal structures of the azurin mutant Met121Ala and its azide derivative Met121Ala-azide from Pseudomonas aeruginosa have been determined. The final crystallographic R values are 21.3 and 19.4% for the two structures, respectively. In the Met121Ala mutant, the distance between the copper ion and His117 increases by 0.34 A compared with the wild-type structure. The removal of the methionine in the apical position induces a shortening of the distance from the copper ion to the carbonyl O atom of Gly45 from 2.97 to 2.74 A. In the Met121Ala-azide structure, the azide anion occupies the cavity created by replacing the Met121 side chain with the smaller methyl group of Ala. The azide anion binds with a terminal N atom to the copper ion at a distance of about 2.04 A. In addition, the copper ion has moved out of the trigonal plane by about 0.26 A towards the azide anion. Thus, the copper site in this structure has a distorted tetrahedral arrangement. The spectroscopic characteristics show, in addition, that the copper sites in the two structures are distinctively different. The Met121Ala mutant still maintains the properties of an ordinary type 1 copper site while the Met121Ala-azide derivative has an absorption maximum at about 409 nm and the copper hyperfine coupling has increased to a value intermediate between those of type 2 copper and the wild-type azurin.

Environment of copper in Pseudomonas aeruginosa azurin probed by binding of exogenous ligands to Met121X (X = Gly, Ala, Val, Leu, or Asp) mutants.

The binding of small exogenous ligands to mutants of the blue copper protein azurin from Pseudomonas aeruginosa, altered in the axial position, Met121X (X = Gly, Ala, Val, Leu, or Asp), has been studied with optical and electron paramagnetic resonance (EPR) spectroscopy. The results show that small molecules can enter the pocket left by the side chain of Met121. For azide, the dissociation constants are Leu > Val > Ala, reflecting the increasing space available. The Gly and Asp mutants bind azide less strongly than the Ala mutant, due to competition with water (Gly) and the polar side chain (Asp). Similar trends are found for thiocyanate. Cyanide binds equally well to the Ala and Val mutants. A number of other small potential ligands were tried. Alcohols do not affect room-temperature optical spectra, but at low temperatures, the EPR spectrum is stellacyanin-like, indicative of a weak axial interaction. Ligands binding with a carboxyl group or nitrogen (e.g. acetate or azide) convert the metal center to a form intermediate between regular types 1 and 2, presumably by pulling the copper ion out of the trigonal plane formed by Cys(S) and two His(N). Cyanide interacts strongly as shown by the hyperfine coupling to the 13C nucleus. With increasing strength of the axial interaction, the two major bands in the visible region (600 and 400-500 nm) shift in parallel to higher energy, and at the same time, the strength of the latter transition increases at the expense of the former. This demonstrates that these transitions have a common origin, namely S-to-Cu charge transfer transition.

Structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa at 2.2 A resolution.

The structure of the azurin mutant nickel-Trp48Met from Pseudomonas aeruginosa has been determined by difference Fourier synthesis using phases from the wild-type azurin model. The final crystallographic R value is 0.170 for 17 394 reflections to a resolution of 2.2 A. The mutant crystallized in the orthorhombic space group P2(1)2(1)2(1), a = 57.4, b = 80.4, c = 110.3 A. The four molecules in the asymmetric unit are packed as a dimer of dimers. The nickel metal site of this mutant structure is similar to the zinc metal site in the azurin Asp47 mutant. The site-specific mutation was performed at residue Trp48, which is located in the center of the protein in a highly hydrophobic environment, to investigate its suggested role in the long-range electron-transfer pathway between the disulfide bond on one side of the protein to the Cu centre. The structure around the mutation site Met48 showed no significant change compared with the wild-type structure.

Disruption of the disulfide bridge in azurin from Pseudomonas aeruginosa.

In the family of small blue-copper proteins azurins are unique in that they contain a disulfide bridge close to the amino-terminal end. It is situated in the 'south' side of the molecule, about 25 A away from the copper. Site-specific mutagenesis was used to exchange one or both of the cysteines in the bridge for serines in Pseudomonas aeruginosa azurin. In the double mutant Cys3Ser-Cys26Ser the Type 1 Cu is converted into Type 2, and the fluorescence of the single internal tryptophan shows that it becomes exposed to a polar environment. The circular dichroism spectrum indicates a loss of beta-structure. Thus, this mutation prevents the correct folding of the protein and the formation of the metal-binding site. Single mutants, Cys3Ser or Cys26Ser, can at least in part form native-like structures as shown by optical, EPR, fluorescence and CD spectroscopy. The Cys3Ser mutant can form a stable intermolecular disulfide bond which promotes the native conformation of the protein.

Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

Intramolecular electron transfer in single-site-mutated azurins.

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)

Reduction potentials and their pH dependence in site-directed-mutant forms of azurin from Pseudomonas aeruginosa.

A spectroelectrochemical method has been used to determine the reduction potential of the copper site in wild-type and 22 mutant forms of azurin from Pseudomonas aeruginosa at 25 degrees C and in the range pH 4-8; the effect of buffers and ionic strength on the potentials has also been studied. Amino-acid residues changed include Met121, which provides an S atom at a distance of about 0.3 nm from the metal, some amino acids in the hydrophobic patch, other residues believed to be important in electron transfer with physiological partners and some internal amino acids. The observed potentials span a range of about 300 mV. In all cases the potentials increase with decreasing pH, but the pKa values describing the pH dependence are essentially unchanged except in three mutants, where they change by pH 0.6-1.1 (up in one and down in two). The largest potential changes were found in some Met121 mutants, at which position large hydrophobic residues raise the potential, whereas negatively charged residues lower it; a decreased potential is also found in the Met121-->End mutant, which probably has H2O coordinated to the metal. Gly45 has its carbonyl group coordinated to copper, but the potential of Gly45-->Ala is close to that of the wild type. Some substitutions in the hydrophobic patch cause an increase in the potential, whereas substitutions involving His35 and Glu91 do not result in significant changes. No single mechanism for tuning the potential of the copper site can be discerned, but in many cases there are probably indirect effects of the protein conformation causing changes in metal-ligand interactions.

Studies of the slowly exchanging chloride in Photosystem II of higher plants.

(36)Cl(-) was used to study the slow exchange of chloride at a binding site associated with Photosystem II (PS II). When PS II membranes were labeled with different concentrations of (36)Cl(-), saturation of binding at about I chloride/PS II was observed. The rate of binding showed a clear dependence on the concentration of chloride approaching a limiting value of about 3·10(-4) s(-1) at high concentrations, similar to the rate of release of chloride from labeled membranes. These rates were close to that found earlier for the release of chloride from PS II membranes isolated from spinach grown on (36)Cl(-), which suggests that we are observing the same site for chloride binding. The similarity between the limiting rate of binding and the rate of release of chloride suggests that the exchange of chloride with the surrounding medium is controlled by an intramolecular process. The binding of chloride showed a pH-dependence with an apparent pKa of 7.5 and was very sensitive to the presence of the extrinsic polypeptides at the PS II donor side. The binding of chloride was competitively inhibited by a few other anions, notably Br(-) and NO3 (-). The slowly exchanging Cl(-) did not show any significant correlation with oxygen evolution rate or yield of EPR signals from the S2 state. Our studies indicate that removal of the slowly exchanging chloride lowers the stability of PS II as indicated by the loss of oxygen evolution activity and S2 state EPR signals.

Modification of the electron-transfer sites of Pseudomonas aeruginosa azurin by site-directed mutagenesis.

Site-directed mutagenesis of the structural gene for azurin from Pseudomonas aeruginosa has been used to prepare azurins in which amino acid residues in two separate electron-transfer sites have been changed: His-35-Lys and Glu-91-Gln at one site and Phe-114-Ala at the other. The charge-transfer band and the EPR spectrum are the same as in the wild-type protein in the first two mutants, whereas in the Phe-114-Ala azurin, the optical band is shifted downwards by 7 nm and the copper hyperfine splitting is decreased by 4.10(-4)/cm. This protein also shows an increase of 20-40 mV in the reduction potential compared to the other azurins. The potentials of all four azurins decrease with increasing pH in phosphate but not in zwitterionic buffers with high ionic strength. The rate constant for electron exchange with cytochrome c551 is unchanged compared to the wild-type protein in the Phe-114-Ala azurin, but is increased in the other two mutant proteins. The results suggest that Glu-91 is not important for the interaction with cytochrome c551 and that His-35 plays no critical role in the electron transfer to the copper site.

The Origin of the Multiline and g = 4.1 Electron Paramagnetic Resonance Signals from the Oxygen-Evolving System of Photosystem II.

Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).

Electron-paramagnetic-resonance studies of structure and function of the two-haem enzymes Pseudomonas cytochrome c peroxidase and beef heart cytochrome c oxidase.

Beef heart cytochrome c oxidase contains two cytochromes, a and a3, and Pseudomonas aeruginosa cytochrome c peroxidase has one high- and one low-potential c haem, cHP and cLP. The parallelism in co-ordination and spin states between cytochrome a and haem cHP on the one hand and between cytochrome a3 and haem cLP on the other is illustrated. The two latter haems become accessible to cyanide, when the former are reduced. Such reduction also leads to an activation of the enzymes. Mechanisms are presented in which ferryl forms of cytochromes a3 and haem cLP take part. The enzymes reach an oxidation state, formally the same as resting enzyme, but with different properties.

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase.

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (Rönnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase.

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.

An investigation by e.p.r. and optical spectroscopy of cytochrome oxidase during turnover.

Cytochrome oxidase (EC 1.9.3.1; ferrocytochrome c:oxygen oxidoreductase) was studied during steady-state by optical and e.p.r. methods. Starting with either the 'resting' or the 'pulsed' enzyme, oxidase, cytochrome c, ascorbate and O2 were mixed and the reaction monitored optically. Tetramethylphenylenediamine was used as mediator to poise the steady-state to the desired reduction level. After mixing, the reaction was quenched by the used of rapid-freeze techniques. The e.p.r. spectra of samples captured at increasing tetramethylphenylenediamine concentrations (i.e. higher electron flux) show decreasing g = 2 (Cu A) and g = 3 (cytochrome a) signals. No Cu B or g = 6 signals (high-spin cytochrome a3) could be found during the reaction. Also, the signal with peaks at g = 1.69, 1.78 and 5 as well as the g = 12 signal was hardly detectable at higher turnover rates. The only new signal appearing during turnover is a radical signal, which is discussed in terms of a protein radical. Finally, a scheme is presented, proposing a catalytic cycle for cytochrome oxidase with respect to the O2 binding Cu B-cytochrome a3 unit.

The structure of the paramagnetic oxygen intermediate in the cytochrome c oxidase reaction.

A paramagnetic intermediate with an unusual e.p.r. spectrum is formed when fully reduced cytochrome c oxidase is allowed to react with dioxygen at 173 K. The effect on the e.p.r. spectrum of using dioxygen enriched in 17O was investigated. These experiments show that an oxygen atom derived from dioxygen is bound to Cu2+ in the intermediate. The e.p.r. parameters can be explained in terms of a weak antiferromagnetic interaction (J approximately equal to 10 cm-1) between Cu2+B and cytochrome a3 in the low-spin ferryl ion state. It is suggested that an OH- ion bound to Cu2+B is hydrogen bonded to the oxygen atom of the ferryl ion in cytochrome a3.

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase.

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.

A new copper(II) electron paramagnetic resonance signal in two laccases and in cytochrome c oxidase.

A new EPR signal from Cu2+ has been discovered in reductive experiments with type 2 copper-depleted laccase from Polyporus versicolor. A novel EPR signal has also been found in native laccase from Rhus vernicifera on oxidation of the reduced protein with H2O2. In reoxidation experiments with cytochrome c oxidase from beef heart, a new Cu2+ signal has been observed. With Rhus laccase, the new signal is shown to originate from one of the copper ions that are nondetectable in the resting enzyme, and evidence is presented for the signals in Polyporus laccase and cytochrome c oxidase also stemming from the metal pairs that are antiferromagnetically coupled in the oxidized enzymes. The new signals show strong rhombic character, and the EPR parameters place them in a category different from the signals of type 1 as well as of type 2 Cu2+ ions.