Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6

AbstractKinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the Cu^(II(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^(II)(Cys112Asp) azurin can be reduced by excess [Ru^(II)(NH_3)_6]^(2+), resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c_(551) (μ = 0.1 M sodium phosphate (pH 7.0);E°(Cu^(II/I)) = 180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^(II)(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^(II)–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group

Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in <it>Heterosigma akashiwo </it>(Raphidophyceae)

<p>Abstract</p> <p>Background</p> <p>Maintenance of homeostasis requires that an organism perceive selected physical and chemical signals within an informationally dense environment. Functionally, an organism uses a variety of signal transduction arrays to amplify and convert these perceived signals into appropriate gene transcriptional responses. These changes in gene expression serve to modify selective metabolic processes and thus optimize reproductive success. Here we analyze a chloroplast-encoded His-to-Asp signal transduction circuit in the stramenopile <it>Heterosigma akashiwo </it>(Hada) Hada <it>ex </it>Y. Hara <it>et </it>Chihara [syn. <it>H. carterae </it>(Hulburt) F.J.R. Taylor]. The presence, structure and putative function of this protein pair are discussed in the context of their evolutionary homologues.</p> <p>Results</p> <p>Bioinformatic analysis of the <it>Heterosigma akashiwo </it>chloroplast genome sequence revealed the presence of a single two-component His-to-Asp (designated Tsg1/Trg1) pair in this stramenopile (golden-brown alga). These data represent the first documentation of a His-to-Asp array in stramenopiles and counter previous reports suggesting that such regulatory proteins are lacking in this taxonomic cluster. Comparison of the 43 kDa <it>H. akashiwo </it>Tsg1 with bacterial sensor kinases showed that the algal protein exhibits a moderately maintained PAS motif in the sensor kinase domain as well as highly conserved H, N, G₁and F motifs within the histidine kinase ATP binding site. Molecular modelling of the 27 kDa <it>H. akashiwo </it>Trg1 regulator protein was consistent with a winged helix-turn-helix identity – a class of proteins that is known to impact gene expression at the level of transcription. The occurrence of Trg1 protein in actively growing <it>H. akashiwo </it>cells was verified by Western analysis. The presence of a PhoB-like RNA polymerase loop in Trg1 and its homologues in the red-algal lineage support the hypothesis that Trg1 and its homologues interact with a sigma 70 (σ⁷⁰) subunit (encoded by <it>rpoD</it>) of a eubacterial type polymerase. Sequence analysis of <it>H. akashiwo rpoD </it>showed this nuclear-encoded gene has a well-defined 4.2 domain, a region that augments RNA polymerase interaction with transcriptional regulatory proteins and also serves in -35 promoter recognition. The presence/loss of the His-to-Asp pairs in primary and secondary chloroplast lineages is assessed.</p> <p>Conclusion</p> <p>His-to-Asp signal transduction components are found in most rhodophytic chloroplasts, as well as in their putative cyanobacterial progenitors. The evolutionary conservation of these proteins argues that they are important for the maintenance of chloroplast homeostasis. Our data suggest that chloroplast gene transcription may be impacted by the interaction of the His-to-Asp regulator protein (which is less frequently lost than the sensor protein) with the RNA polymerase σ⁷⁰subunit.</p

Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in (Raphidophyceae)-3

<p><b>Copyright information:</b></p><p>Taken from "Chloroplast His-to-Asp signal transduction: a potential mechanism for plastid gene regulation in (Raphidophyceae)"</p><p>http://www.biomedcentral.com/1471-2148/7/70</p><p>BMC Evolutionary Biology 2007;7():70-70.</p><p>Published online 3 May 2007</p><p>PMCID:PMC1885438.</p><p></p>f OmpR (grey), PhoB (white), and the complete receiver-regulator structure from (blue) reveals important similarities. The predicted Trg1 model closely resembles that of OmpR, particularly in the putative DNA binding region (α3 helix). Notably, the predicted Trg1 structure for the putative RNA polymerase interaction site (α-αloop, red) more closely matches that of PhoB. The phosphorylation site is shown as a purple sphere