Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Azurin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1992

Characterization and crystal structure of zinc azurin, a by-product of heterologous expression in Escherichia coli of Pseudomonas aeruginosa copper azurin.

Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copper ions. An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45. The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, which is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.

Azurin↗
PubMed1992

Room-temperature phosphorescence from azurin derivatives. Phosphorescence quenching in oxidized native azurin.

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin has been monitored at 30 degrees C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of ca 130 ms is observed from solutions of oxidized native azurin, but this component is ascribed to a modified form of azurin in solution, i.e. protein heterogeneity, on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center.

Azurin↗
PubMed1986

Studies of thermally induced denaturation of azurin and azurin derivatives by differential scanning calorimetry: evidence for copper selectivity.

Azurin, a blue copper protein from Pseudomonas aeruginosa, and several derivatives of azurin have been studied by differential scanning calorimetry. Two well-separated, irreversible transitions are observed in a scan of apoazurin under a variety of conditions, and they are assigned to distinct steps in the denaturation process. No specific structural component can be assigned to the lower temperature transition, but a "flap" structure which is found near the metal binding site may be involved. Circular dichroic spectra suggest that melting of the beta-sheet structure, the main structural motif in the native protein, occurs during the second transition. With the exceptions of the Ni(II) and p-(hydroxymercuri)benzoate derivatives, the transitions are superposed in the metalated forms, and the enthalpies of denaturation are more endothermic. By comparison with other first-row divalent transition ions and especially Zn(II), the Cu(II) derivative exhibits the most endothermic denaturation process. Along with other data, this suggests that the binding energy is greater for Cu(II). It is postulated that the selectivity for copper over zinc arises because of the irregular binding geometry offered by the folded protein. Denaturation of the Hg(II) derivative is even more endothermic, confirming that the type 1 binding site has a very great affinity for Hg(II). Finally, when substoichiometric amounts of Hg(II) are added to the apoprotein, there is evidence that a novel mercury-bridged dimer of azurin forms.

Azurin↗
PubMed1990

Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli.

The azurin-encoding azu gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. A purification procedure was developed to isolate the azurin obtained from the E. coli cells. No differences were observed between azurins isolated from P. aeruginosa and E. coli. A non-reconstitutable azurin-like protein, azurin*, with a spectral ratio (A625/A280) less than 0.01 could be separated from holo-azurin with a spectral ratio of 0.58 (+/- 0.01). The properties of azurin* were examined by electrophoretic (SDS-PAGE and IEF) and spectroscopic (UV/vis, 1H-NMR, static and dynamic fluorescence) techniques, and compared to the properties of holo-azurin and apo-azurin. Azurin* resembles apo-azurin (same pKa* values of His-35 and His-117, same fluorescence characteristics). However, it has lost the ability to bind Cu-ions. It is tentatively concluded that azurin* is a chemically modified form of azurin, the modification possibly being due to oxidation of the ligand residue Cys-112 or the formation of a chemical bond between the ligand residues Cys-112 and His-117. In agreement with previous results from Hutnik and Szabo (Biochemistry (1989) 28, 3923-3934), fluorescence experiments show that the heterogeneous fluorescence decay observed for holo-azurin is not due to the presence of azurin*, but most likely originates from conformational heterogeneity of the holo-azurin.

Azurin↗
PubMed1987

Internal motion and electron transfer in proteins: a picosecond fluorescence study of three homologous azurins.

We have carried out a picosecond fluorescence study of holo- and apoazurins of Pseudomonas aeruginosa (azurin Pae), Alcaligenes faecilis (azurin Afe), and Alcaligenes denitrificans (azurin Ade). Azurin Pae contains a single, buried tryptophyl residue; azurin Afe, a single surface tryptophyl residue; and azurin Ade, tryptophyl residues in both environments. From anisotropy measurements we conclude that the interiors of azurins Pae and Ade are not mobile enough to enable motion of the indole ring on a nanosecond time scale. The exposed tryptophans in azurins Afe and Ade show considerable mobility on a few hundred picosecond time scale. The quenching of tryptophan fluorescence observed in the holoproteins is interpreted in terms of electron transfer from excited-state tryptophan to Cu(II). The observed rates are near the maximum predicted by Marcus theory for the separation of donor and acceptor. The involvement of protein matrix and donor mobility for electron transfer is discussed. The two single-tryptophan-containing proteins enable the more complex fluorescence behavior of the two tryptophans of azurin Ade to be understood. The single-exponential fluorescence decay observed for azurin Pae and the nonexponential fluorescence decay observed for azurin Afe are discussed in terms of current models for tryptophan photophysics.

Alcaligenes↗
PubMed1992

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.

Azurin↗
PubMed1987

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Alcaligenes↗
PubMed1985

Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study.

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

Apoproteins↗
PubMed1989

The azurin gene from Pseudomonas aeruginosa. Cloning and characterization.

We have cloned and sequenced the Pseudomonas aeruginosa azurin structural gene and its flanking regions. The DNA sequence predicts a pre-protein with a signal peptide of 19 amino acids followed by the 128-amino-acid mature azurin protein. Nuclease-S1 mapping and primer elongation experiments indicated two 5' termini of the azurin transcript. The major transcript of the azurin gene is initiated around 35 base pairs upstream from the translational start. The minor transcript, with a promoter region sharing homology with a consensus nif promoter of Klebsiella pneumoniae and also with other Pseudomonas genes, is initiated 145 base pairs upstreams of the azurin initiation codon. Downstream from the azurin structural gene a sequence similar to a transcriptional terminator is found. Northern blot analysis indicated two sizes of the azurin mRNA (0.54 kb and 0.65 kb) confirming the S1 mapping and the predictions from the nucleotide sequence.

Amino Acid Sequence↗
PubMed1992

The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy.

To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.

Alcaligenes↗
PubMed1989

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.

Azurin↗
PubMed1977

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

Azurin↗
PubMed1985

1H NMR studies of electron exchange rate of Pseudomonas aeruginosa azurin.

T1 values of the His-35 C-2 proton resonance of reduced Pseudomonas aeruginosa azurin were determined at 25 degrees C and pH values 4.5, 7.3, and 9.0 in the presence of different fractional amounts of oxidized azurin. The C-2 proton of His-35 undergoes very rapid spin relaxation in oxidized azurin because of its close proximity to the paramagnetic copper. In the presence of oxidized protein, the T1 values of this proton in reduced azurin depend on the lifetime of the reduced protein. From the T1 data, the electron self-exchange rate constant for azurin was calculated to be 1.4 X 10(4) M-1 X s-1, 4.3 X 10(3) M-1 X s-1, and 6.0 X 10(3) M-1 X s-1 at pH values 4.5, 7.3, and 9, respectively. At pH 7.3, the C-2 proton of His-35 is in slow exchange between the imidazole and imidazolium forms and gives rise to two separate resonances at 9.39 and 8.00 ppm. By using these two resonances, the electron self-exchange rate constants were determined separately for the two species of azurin for which the His-35 residue is in the imidazole or the imidazolium forms; results showed that both species participate in self-exchange of electrons with equal efficiency.

Azurin↗
PubMed1992

Complete sequential 1H and 15N nuclear magnetic resonance assignments and solution secondary structure of the blue copper protein azurin from Pseudomonas aeruginosa.

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14,000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40 degrees C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear 1H Hartmann-Hahn nuclear Overhauser (3D 1H HOHAHA-NOESY) spectrum and a 3D heteronuclear 1H nuclear Overhauser 1H[15N] single-quantum coherence (3D 1H[15N] NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40 degrees C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 N epsilon 2H resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed.

Amino Acid Sequence↗
PubMed1976

Conformational equilibria accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa.

The redox reaction between cytochrome c (Cyt c) (P-551) and the blue copper protein azurin, both from Pseudomonas aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve three equilibria. The fast relaxation time (0.4 less than tau less than 8 ms) was ascribed to the electron exchange step. The slow relaxation time (tau congruent to 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). But because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the two slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (tau congruent to 0.1 ms) would be zero. At 25 degrees C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 X 10(6) and 7.8 X 10(6) M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at approximately 10(4) s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35 degrees C), i.e. deltaHtot = 0. This zero enthalpy change was found to be compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were two to three times higher than the values published for various low molecular weight reagents in their electron exchange with copper proteins, yet the rate of exchange between Cyt c (P551) and azurin was some hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer sphere reaction, of which the rate could be approximated by the Marcus theory.

Azurin↗
PubMed1991

Gene synthesis, expression, and mutagenesis of the blue copper proteins azurin and plastocyanin.

Genes for the blue copper proteins Populus nigra var. italica plastocyanin and Pseudomonas aeruginosa azurin have been constructed by a stepwise procedure. The leader sequence for azurin has been placed before the genes directing plastocyanin and azurin transport to the periplasmic space when the genes are expressed in Escherichia coli. Site-saturation mutagenesis has been used to alter two copper-binding residues of azurin (Met-121 and His-46) and Met-92 of plastocyanin. While the plastocyanin mutants do not appear to bind copper, the azurin variants all bind copper and show characteristic type I blue copper centers. In particular, the electronic spectra reflect the dominance of the charge transfer interaction between copper and the thiolate of Cys-112, being relatively insensitive to changes in Met-121 or His-46. In contrast, removal of Met-121 appreciably alters the EPR spectra of the mutants, although, to a first order, the spectra of all mutants are themselves similar, suggesting a more distorted geometry around copper in the mutants than in the wild type.

Amino Acid Sequence↗
PubMed1990

Involvement of the hydrophobic patch of azurin in the electron-transfer reactions with cytochrome C551 and nitrite reductase.

The electron-transfer reactions of site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c551 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch of azurin Met44 was replaced by Lys, and in the His35 patch His35 was replaced by Phe, Leu and Gln. Both patches were previously thought to be involved in electron transfer. 1H-NMR spectroscopy revealed only minor changes in the three-dimensional structure of the mutants compared to wild-type azurin. Observed changes in midpoint potentials could be attributed to electrostatic effects. The slow relaxation phase observed in temperature-jump experiments carried out on equilibrium mixtures of wild-type azurin and cytochrome c551 was definitively shown to be due to a conformational relaxation involving His35. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions with both cytochrome c551 and nitrite reductase.

Amino Acid Sequence↗