The linked conservation of structure and function in a family of high diversity: the monomeric cupredoxins. (57/282)

The monomeric cupredoxins are a highly divergent family of copper binding electron transport proteins that function in photosynthesis and respiration. To determine how function and structure are conserved in the context of large sequence differences, we have carried out a detailed analysis of the cupredoxins of known structure and their sequence homologs. The common structure of the cupredoxins is formed by a sandwich of two beta sheets which support a copper binding site. The structure of the deeply buried core is intimately coupled to the binding site on the surface of the protein; in each protein the conserved regions form one continuous substructure that extends from the surface active site and through the center of the molecule. Residues around the active site are conserved for functional reasons, while those deeper in the structure will be conserved for structural reasons. Together the two sets support each other.  (+info)

pH-dependent transition between delocalized and trapped valence states of a CuA center and its possible role in proton-coupled electron transfer. (58/282)

A pH-dependent transition between delocalized and trapped mixed valence states of an engineered CuA center in azurin has been investigated by UV-visible absorption and electron paramagnetic resonance spectroscopic techniques. At pH 7.0, the CuA azurin displays a typical delocalized mixed valence dinuclear [Cu(1.5)....Cu(1.5)] spectra with optical absorptions at 485, 530, and 760 nm, and with a seven-line EPR hyperfine. Upon lowering of the pH from 7.0 to 4.0, the absorption at 760 nm shifted to lower energy toward 810 nm, and a four-line EPR hyperfine, typical of a trapped valence, was observed. The pH-dependent transition is reversible because increasing the pH restores all delocalized spectral features. Lowering the pH resulted in not only a trapped valence state, but also a dramatically increased reduction potential of the Cu center (from 160 mV to 340 mV). Mutation of the titratable residues around the metal-binding site ruled out Glu-114 and identified the C-terminal histidine ligand (His-120) as a site of protonation, because the His120Ala mutation abolished the above pH-dependent transition. The corresponding histidine in cytochrome c oxidases is along a major electron transfer pathway from CuA center to heme a. Because the protonation of this histidine can result in an increased reduction potential that will prevent electron flow from the CuA to heme a, the CuA and the histidine may play an important role in regulating proton-coupled electron transfer.  (+info)

Probing the influence on folding behavior of structurally conserved core residues in P. aeruginosa apo-azurin. (59/282)

The effects on folding kinetics and equilibrium stability of core mutations in the apo-mutant C112S of azurin from Pseudomonas aeruginosa were studied. A number of conserved residues within the cupredoxin family were recognized by sequential alignment as constituting a common hydrophobic core: I7, F15, L33, W48, F110, L50, V95, and V31. Of these, I7, V31, L33, and L50 were mutated for the purpose of obtaining information on the transition state and a potential folding nucleus. In addition, residue V5 in the immediate vicinity of the common core, as well as T52, separate from the core, were mutated as controls. All mutants exhibited a nonlinear dependence of activation free energy of folding on denaturant concentration, although the refolding kinetics of the V31A/C112S mutant indicated that the V31A mutation destabilizes the transition state enough to allow folding via a parallel transition state ensemble. Phi-values could be calculated for three of the six mutants, V31A/C112S, L33A/C112S, and L50A/C112S, and the fractional values of 0.63, 0.33, and 0.50 (respectively) obtained at 0.5 M GdmCl suggest that these residues are important for stabilizing the transition state. Furthermore, a linear dependence of ln k(obs)(H2O) on DeltaG(U-N)(H2O) of the core mutations and the putative involvement of ground-state effects suggest the presence of native-like residual interactions in the denatured state that bias this ensemble toward a folding-competent state.  (+info)

Conformational changes in azurin from Pseudomona aeruginosa induced through chemical and physical protocols. (60/282)

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.  (+info)

Functionally specified protein signatures distinctive for each of the different blue copper proteins. (61/282)

BACKGROUND: Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands. RESULTS: Sequences that share such conserved motifs are crucial to the structure or function of the protein and this could provide a signature of family membership. The blue copper proteins chosen for the study were plantacyanin, plastocyanin, cucumber basic protein, stellacyanin, dicyanin, umecyanin, uclacyanin, cusacyanin, rusticyanin, sulfocyanin, halocyanin, azurin, pseudoazurin, amicyanin and nitrite reductase which were identified in both eukaryotes and prokaryotes. ClustalW analysis of the protein sequences of each of the blue copper proteins was the basis for designing protein signatures or peptides. The protein signatures and peptides identified in this study were designed involving the active site region involving the amino acids bound to the copper atom. It was highly specific for each kind of blue copper protein and the false picks were minimized. The set of signatures designed specifically for the BCP's was entirely different from the existing broad spectrum signatures as mentioned in the background section. CONCLUSIONS: These signatures can be very useful for the annotation of uncharacterized proteins and highly specific to retrieve blue copper protein sequences of interest from the non redundant databases containing a large deposition of protein sequences.  (+info)

Apo-azurin folds via an intermediate that resembles the molten-globule. (62/282)

The folding of Pseudomonas aeruginosa apo-azurin was investigated with the intent of identifying putative intermediates. Two apo-mutants were constructed by replacing the main metal-binding ligand C112 with a serine (C112S) and an alanine (C112A). The guanidinium-induced unfolding free energies (DeltaG(U-N)(H2O)) of the C112S and C112A mutants were measured to 36.8 +/- 1 kJ mole(-1) and 26.1 +/- 1 kJ mole(-1), respectively, and the m-value of the transition to 23.5 +/- 0.7 kJ mole(-1) M(-1). The difference in folding free energy (DeltaDeltaG(U-N)(H2O)) is largely attributed to the intramolecular hydrogen bonding properties of the serine Ogamma in the C112S mutant, which is lacking in the C112A structure. Furthermore, only the unfolding rates differ between the two mutants, thus pointing to the energy of the native state as the source of the observed Delta DeltaG(U-N)(H2O). This also indicates that the formation of the hydrogen bonds present in C112S but absent in C112A is a late event in the folding of the apo-protein, thus suggesting that formation of the metal-binding site occurs after the rate-limiting formation of the transition state. In both mutants we also noted a burst-phase intermediate. Because this intermediate was capable of binding 1-anilinonaphtalene-8-sulfonate (ANS), as were an acid-induced species at pH 2.6, we ascribe it molten globule-like status. However, despite the presence of an intermediate, the folding of apo-azurin C112S is well approximated by a two-state kinetic mechanism.  (+info)

Rusticyanin, a bacterial electron transfer protein, causes G1 arrest in J774 and apoptosis in human cancer cells. (63/282)

During acid mine drainage, Acidithiobacillus ferrooxidans, a nonpathogenic, acidophilic, lithotrophic bacterium, utilizes rusticyanin to transfer electrons for the oxidation of Fe(2+) to Fe(3+) for deriving its energy. No other function of rusticyanin is known. We demonstrate that purified rusticyanin enters mammalian cells inducing either inhibition of cell cycle progression or caspase-8 mediated apoptosis. Treatment of human melanoma cells with rusticyanin allowed significant generation of reactive oxygen species and active caspase-8, leading to cell death. The ability of rusticyanin to modulate mammalian cell death might be relevant to a role of this cupredoxin in protecting At. ferrooxidans from eukaryotic predators in the environment.  (+info)

Structure-based engineering of Alcaligenes xylosoxidans copper-containing nitrite reductase enhances intermolecular electron transfer reaction with pseudoazurin. (64/282)

The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.87 x 10(5) M(-1) s(-1)) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K(m) = 20 microM and k(ET) = 7.3 x 10(5) M(-1) s(-1)). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The k(ET) values of these mutants become progressively larger as the number of mutated residues increases. The K(m) and k(ET) values of A83D/A191E/G198E (K(m) = 88 microM and k(ET) = 4.1 x 10(5) M(-1) s(-1)) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.  (+info)