Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin

Marks, Jessica R.; Pozdnyakova, Irina; Guidry, Jessie; Wittung-Stafshede, Pernilla

doi:10.1007/s00775-004-0523-6

Cited by 35 publications

(59 citation statements)

References 22 publications

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“…Overall, the designed constructs bind copper with 1 to 5 orders of magnitude weaker affinity than azurin, which has corresponding values of 0.03 and 25 fM for Cu(I) and Cu(II) at pH 7.0. 35 …”

Section: Resultsmentioning

confidence: 99%

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

Plegaria¹,

Duca

Tard

et al. 2015

Inorg. Chem.

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Using de novo protein design, we incorporated a copper metal binding site within the three-helix bundle α3D (Walsh et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5486–5491) to assess whether a cupredoxin center within an α-helical domain could mimic the spectroscopic, structural, and redox features of native type-1 copper (CuT1) proteins. We aimed to determine whether a CuT1 center could be realized in a markedly different scaffold rather than the native β-barrel fold and whether the characteristic short Cu–S bond (2.1–2.2 Å) and positive reduction potentials could be decoupled from the spectroscopic properties (ε600 nm = 5000 M−1 cm−1) of such centers. We incorporated 2HisCys(Met) residues in three distinct α3D designs designated core (CR), chelate (CH), and chelate-core (ChC). XAS analysis revealed a coordination environment similar to reduced CuT1 proteins, producing Cu–S(Cys) bonds ranging from 2.16 to 2.23 Å and Cu–N(His) bond distances of 1.92–1.99 Å. However, Cu(II) binding to the CR and CH constructs resulted in tetragonal type-2 copper-like species, displaying an intense absorption band between 380 and 400 nm (>1500 M−1 cm−1) and A∥ values of (150–185) × 10−4 cm−4. The ChC construct, which possesses a metal-binding site deeper in its helical bundle, yielded a CuT1-like brown copper species, with two absorption bands at 401 (4429 M−1 cm−1) and 499 (2020 M−1 cm−1) nm and an A∥ value ~30 × 10−4 cm−4 greater than its native counterparts. Electrochemical studies demonstrated reduction potentials of +360 to +460 mV (vs NHE), which are within the observed range for azurin and plastocyanin. These observations showed that the designed metal binding sites lacked the necessary rigidity to enforce the appropriate structural constraints for a Cu(II) chromophore (EPR and UV–vis); however, the Cu(I) structural environment and the high positive potential of CuT1 centers were recapitulated within the α-helical bundle of α3D.

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Section: Resultsmentioning

confidence: 99%

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

Plegaria¹,

Duca

Tard

et al. 2015

Inorg. Chem.

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“…55 Protein degradation is also possible. In the case of Cu I AzFFF, the increase in Cu II signal is attributed to path C in which an electron is ejected directly from the Cu I center.…”

Section: Discussionmentioning

confidence: 99%

Photogeneration and Quenching of Tryptophan Radical in Azurin

et al. 2015

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Tryptophan and tyrosine can form radical intermediates that enable long-range, multistep electron transfer (ET) reactions in proteins. This report describes the mechanisms of formation and quenching of a neutral tryptophan radical in azurin, a blue-copper protein that contains native tyrosine (Y108 and Y72) and tryptophan (W48) residues. A long-lived neutral tryptophan radical W48• is formed upon UV-photoexcitation of a zinc(II)-substituted azurin mutant in the presence of an external electron acceptor. The quantum yield of W48• formation (Φ) depends upon the tyrosine residues in the protein. A tyrosine-deficient mutant, ZnIIAz48W, exhibited a value of Φ = 0.080 with a Co(III) electron acceptor. A nearly identical quantum yield was observed when the electron acceptor was the analogous tyrosine-free, copper(II) mutant; this result for the ZnIIAz48W:CuIIAz48W mixture suggests there is an interprotein ET path. A single tyrosine residue at one of the native positions reduced the quantum yield to 0.062 (Y108) or 0.067 (Y72). Wild-type azurin with two tyrosine residues exhibited a quantum yield of Φ = 0.045. These data indicate that tyrosine is able to quench the tryptophan radical in azurin.

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“…Considering that the resolution provided by x-ray crystallography for the Cu 2ϩ -and Zn 2ϩ -metallated azurin structures is presently limited to 1.5 Å, the thioether's sulfur interactions with the cofactor are geometrically indistinguishable in practice. Moreover, the role of M121 as a coordinating residue in the unfolded state is still not settled (10,15,20,32). Accordingly, this particular residue was not explicitly modeled as a coordinating residue, only as a contacting residue.…”

Section: The Theoretical Foundationmentioning

confidence: 99%

“…Upon unfolding of metallated azurin, the copper remains bound to the denatured polypeptide in a trigonal coordination composed of the native ligands C112, H117, and possibly M121 (15). In the denatured state the slow irreversible redox coupling between the C112 thiol and Cu 2ϩ promotes sulfur oxidation.…”

mentioning

confidence: 99%

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

Zong

Wilson

Shen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Understanding how the folding of proteins establishes their functional characteristics at the molecular level challenges both theorists and experimentalists. The simplest test beds for confronting this issue are provided by electron transfer proteins. The environment provided by the folded protein to the cofactor tunes the metal's electron transport capabilities as envisioned in the entatic hypothesis. To see how the entatic state is achieved one must study how the folding landscape affects and in turn is affected by the metal. Here, we develop a coarse-grained functional to explicitly model how the coordination of the metal (which results in a so-called entatic or rack-induced state) modifies the folding of the metallated Pseudomonas aeruginosa azurin. Our free-energy functional-based approach directly yields the proper nonlinear extrathermodynamic free energy relationships for the kinetics of folding the wild type and several point-mutated variants of the metallated protein. The results agree quite well with corresponding laboratory experiments. Moreover, our modified free-energy functional provides a sufficient level of detail to explicitly model how the geometric entatic state of the metal modifies the dynamic folding nucleus of azurin.curved chevron ͉ cupredoxin ͉ metalloproteins T he entatic state occurs in proteins when a group, metal or nonmetal, is forced into an unusual, energetically strained geometric or electronic state (rack-induced state) (1-4). Through the polypeptide's folding-induced rigidity, the protein fails to provide the expected geometry of ligating groups that would occur with freely mobile ligands in solution, thereby tuning the ligand's redox characteristics. In metalloproteins, the metal ions are typically bound to the protein through one or more lone pair donors, endogenous biological ligands (e.g., the imidazole moiety of histidine, the carbonyl oxygen of the main chain or the side chain of an asparagine residue). In several cases the ligands are arranged such that an optimal geometry is precluded (1-4). The resulting entatic state in a given metalloprotein is determined by the entire rigid protein scaffold in concert with the hydrogen-bonding network proximal to the coordination sphere (5, 6). The particular geometry of the rack-induced state influences the electronic structure of the metal site. Moreover, the resulting forced electronic structure, at least in certain cases, becomes essential for the protein's biochemical function in electron transport (7). We should remember the entatic hypothesis is in some respects still controversial. Results from some quantum calculations have suggested that the geometry of metal-ligand complexes identified as being rack-induced are not necessarily highly strained (8), whereas other theoretical studies suggest that the rigidity of the protein may in fact be much more significant than initially thought (9).Cupredoxins, a family of electron-transfer metalloproteins, are believed to adopt such a rack-induced state by way of a distorted tetrahedra...

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Methionine-121 coordination determines metal specificity in unfolded Pseudomonas aeruginosa azurin

Cited by 35 publications

References 22 publications

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

Photogeneration and Quenching of Tryptophan Radical in Azurin

Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin

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