Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin

We have used low-temperature (77 K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak π–π interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For thi...

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Bibliographic Details
Published in:Journal of inorganic biochemistry 2010-03, Vol.104 (3), p.250-260
Main Authors: Fitzpatrick, Marzena B., Obara, Yuji, Fujita, Koyu, Brown, Doreen E., Dooley, David M., Kohzuma, Takamitsu, Czernuszewicz, Roman S.
Format: Article
Language:English
Subjects:
Achromobacter cycloclastes - chemistry
Achromobacter cycloclastes - genetics
Azurin - chemistry
Azurin - genetics
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Blue copper
Carrier Proteins - chemistry
Carrier Proteins - genetics
Catalytic Domain
Cupredoxin
Electrochemistry
Metalloproteins - chemistry
Metalloproteins - genetics
Methionine - genetics
Methionine - metabolism
Models, Molecular
Mutation
Non-covalent interaction
Protein Conformation
Pseudoazurin
Resonance Raman
Spectrum Analysis, Raman
Type 1 Cu
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Summary:We have used low-temperature (77 K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak π–π interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin ( AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100–500 cm −1 spectral region. Subtle strengthening of the Cu(II)–S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.3–0.8 cm −1) RR bands involving ν(Cu–S) Cys stretching modes. In contrast, the RR spectra of Met16 mutants with aliphatic amino acids revealed larger (0.2–1.8 cm −1) shifts of the ν(Cu–S) Cys stretching modes to a lower frequency region, which indicate a weakening of the Cu(II)–S(Cys) bond. Comparisons of the predominantly ν(Cu–S) Cys stretching RR peaks of the Met16X = Tyr, Trp, and Phe variants, with the molar absorptivity ratio ε 1/ ε 2 of σ(∼455 nm)/π(∼595 nm) (Cys)S → Cu(II) charge-transfer bands in the optical spectrum and the axial/rhombic EPR signals, revealed a slightly more trigonal disposition of ligands about the copper(II) ion. In contrast, the RR spectra of Met16Z = Ala, Val, Leu, and Ile variants with aliphatic amino acid side chains show a more tetrahedral perturbation of the copper active site, as judged by the lower frequencies of the ν(Cu–S) Cys stretching modes, much larger values of the ε 1/ ε 2 ratio, and the increased rhombicity of the EPR spectra.
ISSN:0162-0134
1873-3344
DOI:10.1016/j.jinorgbio.2009.11.004