A new type 2 copper cysteinate azurin. Involvement of an engineered exposed cysteine in copper binding through internal rearrangement.
(33/282)The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell. (+info)
Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer.
(34/282)The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment. (+info)
If space is provided, bulky modification on the rim of azurin's beta-barrel results in folded protein.
(35/282)Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided. (+info)
The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53.
(36/282)Two redox proteins, azurin and cytochrome c(551) elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein. (+info)
NMR detection of multiple transitions to low-populated states in azurin.
(37/282)Transitions to conformational states with very low populations were detected for the reduced blue copper protein azurin from Pseudomonas aeruginosa by applying constant relaxation time CPMG measurements to the backbone (15)N nuclei at three magnetic fields (11.7, 14.1, and 18.8 T) and three temperatures (25.7, 35.4, and 44.8 degrees C). Two exchange processes with different rate constants could be discriminated despite populations of the excited states below 1% and spatial neighborhood of the two processes. The group of (15)N nuclei involved in the faster process exhibits at 44.8 degrees C a forward rate constant of 11.7+/-2.4 s(-1) and a population of the exited state of 0.39+/-0.07%. They surround the aromatic ring of histidine 35 whose protonation state is coupled to the flipping of a neighboring peptide plane. For the slower process, the forward rate constant and population of the exited state at 44.8 degrees C are 4.1+/-0.1 s(-1) and 0.45+/-0.02%, respectively. The residues involved cluster nearby the copper ion, which is separated from the protonation site of histidine 35 by about 8 A, indicating conformational rearrangements involving the copper coordinating loops. The dependence of the equilibrium constant on the temperature is consistent with an enthalpy-dominated transition around the copper, but an entropy-controlled transition near histidine 35. The detection by nuclear magnetic resonance of millisecond to second conformational transitions near the copper ion suggests a low energy-cost rearrangement of the copper-binding site that may be necessary for efficient electron transfer. (+info)
Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa.
(38/282)Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction. (+info)
Energy-generating enzymes of Burkholderia cepacia and their interactions with macrophages.
(39/282)We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secreted ATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38 was shown to be greatly enhanced in the presence of alpha(2)-macroglobulin. Fractionation of the growth medium of cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additional proteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c(551), which are normally involved in electron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium of B. cepacia strain 71 enriched with the azurin and cytochrome c(551) homologues triggered apoptosis in macrophages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction with antiazurin and anti-cytochrome c(551) antibodies greatly reduced cell death. We cloned and hyperexpressed a gene from B. cepacia strain 71 that encodes the homologue of P. aeruginosa azurin. Such azurin homologues were detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in the growth medium of strains belonging to other genomovars. The growth medium of the strains that elaborated the azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown to induce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol and nucleus when incubated with or microinjected into macrophages. This is an interesting example of the interaction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death. (+info)
The structure of Acidithiobacillus ferrooxidans c(4)-cytochrome: a model for complex-induced electron transfer tuning.
(40/282)The study of electron transfer between the copper protein rusticyanin (RCy) and the c(4)-cytochrome CYC(41) of the acidophilic bacterium Acidithiobacillus ferrooxidans has evidenced a remarkable decrease of RCy's redox potential upon complex formation. The structure of the CYC(41) obtained at 2.2 A resolution highlighted a specific glutamate residue (E121) involved in zinc binding as potentially playing a central role in this effect, required for the electron transfer to occur. EPR and stopped-flow experiments confirmed the strong inhibitory effect of divalent cations on CYC(41):RCy complex formation. A docking analysis of the CYC(41) and RCy structure allows us to propose a detailed model for the complex-induced tuning of electron transfer in agreement with our experimental data, which could be representative of other copper proteins involved in electron transfer. (+info)