Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin

Michael Hay, Marjorie C. Ang, Daniel R. Camelin, Edward I. Solomon, William E. Antholine, Martina Ralle, Ninian J. Blackburn, Priscilla D. Massey, Xiaotang Wang, Angela H. Kwon, Yi Lu

Research output: Contribution to journalArticle

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Abstract

Spectroscopic characterization of a purple CuA center engineered into the blue copper protein azurin from Pseudomonas aeruginosa (called purple CuA azurin hereafter) is presented. Both electrospray mass spectrometry and copper analysis indicated the protein binds two copper ions per protein. The electronic absorption (UV-vis) magnetic circular dichroism (MCD). multifrequency electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectra of the purple CuA azurin are strikingly similar to other native or engineered CuA centers, indicating that they all share similar geometric and electronic structures. It has the characteristic UV-vis absorption spectrum of a CuA center with absorption bands at 485 (ε = 3730), 530 (ε = 3370), 360 (ε = 550), and 770 nm (ε = 1640 M-1 cm-1) The MCD spectrum of purple CuA azurin is dominated by a pair of intense, oppositely-signed features occurring at 480 nm (Δε = -118 deg M-1 cm-1 T-1) and 530 nm (Δε = 155 deg M-1 cm-1 T-1) and a negative feature occurring at 810 nm (Δε = -52 deg M-1 cm-1 T-1). Multifrequency EPR spectra show a well-resolved seven-line hyperfine structure in the g region, typical of a delocalized mixed-valence [Cu(1.5)⋯ Cu(1.5)] binuclear center. Compared with other delocalized mixed-valence CuA centers, this purple CuA azurin has a relatively high energy near-IR Cu-Cu σ → σ* absorption at 770 nm. the largest A→ at 55 G, and the shortest Cu-Cu distance at 2.39 Å. These results may reflect a more sterically compressed CuA center in azurin, perhaps as the result of forcing the normally mononuclear blue copper center in azurin to accept a binuclear CuA center. and are consistent with the general trend between the near-IR Cu-Cu σ → σ* absorption and the degree of Cu2(SR)2 core contraction.

Original languageEnglish (US)
Pages (from-to)191-198
Number of pages8
JournalInorganic Chemistry
Volume37
Issue number2
StatePublished - Dec 1 1998

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Azurin
Copper
Absorption spectra
copper
Paramagnetic resonance
proteins
absorption spectra
dichroism
electron paramagnetic resonance
Proteins
valence
X ray absorption
pseudomonas
Electronic structure
Mass spectrometry
hyperfine structure
contraction
Ions
mass spectroscopy
electronic structure

All Science Journal Classification (ASJC) codes

  • Physical and Theoretical Chemistry
  • Inorganic Chemistry

Cite this

Hay, M., Ang, M. C., Camelin, D. R., Solomon, E. I., Antholine, W. E., Ralle, M., ... Lu, Y. (1998). Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin. Inorganic Chemistry, 37(2), 191-198.
Hay, Michael ; Ang, Marjorie C. ; Camelin, Daniel R. ; Solomon, Edward I. ; Antholine, William E. ; Ralle, Martina ; Blackburn, Ninian J. ; Massey, Priscilla D. ; Wang, Xiaotang ; Kwon, Angela H. ; Lu, Yi. / Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin. In: Inorganic Chemistry. 1998 ; Vol. 37, No. 2. pp. 191-198.
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abstract = "Spectroscopic characterization of a purple CuA center engineered into the blue copper protein azurin from Pseudomonas aeruginosa (called purple CuA azurin hereafter) is presented. Both electrospray mass spectrometry and copper analysis indicated the protein binds two copper ions per protein. The electronic absorption (UV-vis) magnetic circular dichroism (MCD). multifrequency electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectra of the purple CuA azurin are strikingly similar to other native or engineered CuA centers, indicating that they all share similar geometric and electronic structures. It has the characteristic UV-vis absorption spectrum of a CuA center with absorption bands at 485 (ε = 3730), 530 (ε = 3370), 360 (ε = 550), and 770 nm (ε = 1640 M-1 cm-1) The MCD spectrum of purple CuA azurin is dominated by a pair of intense, oppositely-signed features occurring at 480 nm (Δε = -118 deg M-1 cm-1 T-1) and 530 nm (Δε = 155 deg M-1 cm-1 T-1) and a negative feature occurring at 810 nm (Δε = -52 deg M-1 cm-1 T-1). Multifrequency EPR spectra show a well-resolved seven-line hyperfine structure in the g∥ region, typical of a delocalized mixed-valence [Cu(1.5)⋯ Cu(1.5)] binuclear center. Compared with other delocalized mixed-valence CuA centers, this purple CuA azurin has a relatively high energy near-IR Cu-Cu σ → σ* absorption at 770 nm. the largest A→ at 55 G, and the shortest Cu-Cu distance at 2.39 {\AA}. These results may reflect a more sterically compressed CuA center in azurin, perhaps as the result of forcing the normally mononuclear blue copper center in azurin to accept a binuclear CuA center. and are consistent with the general trend between the near-IR Cu-Cu σ → σ* absorption and the degree of Cu2(SR)2 core contraction.",
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Hay, M, Ang, MC, Camelin, DR, Solomon, EI, Antholine, WE, Ralle, M, Blackburn, NJ, Massey, PD, Wang, X, Kwon, AH & Lu, Y 1998, 'Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin', Inorganic Chemistry, vol. 37, no. 2, pp. 191-198.

Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin. / Hay, Michael; Ang, Marjorie C.; Camelin, Daniel R.; Solomon, Edward I.; Antholine, William E.; Ralle, Martina; Blackburn, Ninian J.; Massey, Priscilla D.; Wang, Xiaotang; Kwon, Angela H.; Lu, Yi.

In: Inorganic Chemistry, Vol. 37, No. 2, 01.12.1998, p. 191-198.

Research output: Contribution to journalArticle

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T1 - Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin

AU - Hay, Michael

AU - Ang, Marjorie C.

AU - Camelin, Daniel R.

AU - Solomon, Edward I.

AU - Antholine, William E.

AU - Ralle, Martina

AU - Blackburn, Ninian J.

AU - Massey, Priscilla D.

AU - Wang, Xiaotang

AU - Kwon, Angela H.

AU - Lu, Yi

PY - 1998/12/1

Y1 - 1998/12/1

N2 - Spectroscopic characterization of a purple CuA center engineered into the blue copper protein azurin from Pseudomonas aeruginosa (called purple CuA azurin hereafter) is presented. Both electrospray mass spectrometry and copper analysis indicated the protein binds two copper ions per protein. The electronic absorption (UV-vis) magnetic circular dichroism (MCD). multifrequency electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectra of the purple CuA azurin are strikingly similar to other native or engineered CuA centers, indicating that they all share similar geometric and electronic structures. It has the characteristic UV-vis absorption spectrum of a CuA center with absorption bands at 485 (ε = 3730), 530 (ε = 3370), 360 (ε = 550), and 770 nm (ε = 1640 M-1 cm-1) The MCD spectrum of purple CuA azurin is dominated by a pair of intense, oppositely-signed features occurring at 480 nm (Δε = -118 deg M-1 cm-1 T-1) and 530 nm (Δε = 155 deg M-1 cm-1 T-1) and a negative feature occurring at 810 nm (Δε = -52 deg M-1 cm-1 T-1). Multifrequency EPR spectra show a well-resolved seven-line hyperfine structure in the g∥ region, typical of a delocalized mixed-valence [Cu(1.5)⋯ Cu(1.5)] binuclear center. Compared with other delocalized mixed-valence CuA centers, this purple CuA azurin has a relatively high energy near-IR Cu-Cu σ → σ* absorption at 770 nm. the largest A→ at 55 G, and the shortest Cu-Cu distance at 2.39 Å. These results may reflect a more sterically compressed CuA center in azurin, perhaps as the result of forcing the normally mononuclear blue copper center in azurin to accept a binuclear CuA center. and are consistent with the general trend between the near-IR Cu-Cu σ → σ* absorption and the degree of Cu2(SR)2 core contraction.

AB - Spectroscopic characterization of a purple CuA center engineered into the blue copper protein azurin from Pseudomonas aeruginosa (called purple CuA azurin hereafter) is presented. Both electrospray mass spectrometry and copper analysis indicated the protein binds two copper ions per protein. The electronic absorption (UV-vis) magnetic circular dichroism (MCD). multifrequency electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectra of the purple CuA azurin are strikingly similar to other native or engineered CuA centers, indicating that they all share similar geometric and electronic structures. It has the characteristic UV-vis absorption spectrum of a CuA center with absorption bands at 485 (ε = 3730), 530 (ε = 3370), 360 (ε = 550), and 770 nm (ε = 1640 M-1 cm-1) The MCD spectrum of purple CuA azurin is dominated by a pair of intense, oppositely-signed features occurring at 480 nm (Δε = -118 deg M-1 cm-1 T-1) and 530 nm (Δε = 155 deg M-1 cm-1 T-1) and a negative feature occurring at 810 nm (Δε = -52 deg M-1 cm-1 T-1). Multifrequency EPR spectra show a well-resolved seven-line hyperfine structure in the g∥ region, typical of a delocalized mixed-valence [Cu(1.5)⋯ Cu(1.5)] binuclear center. Compared with other delocalized mixed-valence CuA centers, this purple CuA azurin has a relatively high energy near-IR Cu-Cu σ → σ* absorption at 770 nm. the largest A→ at 55 G, and the shortest Cu-Cu distance at 2.39 Å. These results may reflect a more sterically compressed CuA center in azurin, perhaps as the result of forcing the normally mononuclear blue copper center in azurin to accept a binuclear CuA center. and are consistent with the general trend between the near-IR Cu-Cu σ → σ* absorption and the degree of Cu2(SR)2 core contraction.

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Hay M, Ang MC, Camelin DR, Solomon EI, Antholine WE, Ralle M et al. Spectroscopic Characterization of an Engineered Purple CuA Center in Azurin. Inorganic Chemistry. 1998 Dec 1;37(2):191-198.