Excited-State Charge-Transfer Dynamics of Azurin, a Blue Copper Protein, from Resonance Raman Intensities

Abstract

Resonance Raman spectra of azurin, a 14.6 kDa Type 1 blue copper protein from Pseudomonas aeruginosa, have been measured at wavelengths throughout the S(Cys) → Cu(II) charge-transfer absorption band centered at 625 nm in an effort to determine the role of environment and structure on the dynamics of excited-state charge transfer. Azurin provides an analogous system to plastocyanin, another Type 1 blue copper protein, whose excited-state structure and dynamics have been previously determined for a number of plant species. Self-consistent analysis of the absorption spectrum and the resulting resonance Raman excitation profiles using a time-dependent wave packet propagation formalism indicates that inhomogeneous effects account for the majority of the spectral broadening of the charge-transfer absorption band, in contrast to the primarily homogeneously broadened charge-transfer absorption band in plastocyanin. The total reorganization energy from the resonance Raman enhanced modes was found to be 0.26 ± 0.02 eV, compared to 0.19 ± 0.02 eV for plastocyanin. A detailed comparison of the copper environment in the two proteins reveals specific differences in structure and hydrogen-bonding environment which may explain the differences in observed excited-state charge-transfer dynamics of azurin and plastocyanin. The X-ray crystal structures of poplar a plastocyanin and P. aeruginosa azurin suggest that the larger coordination number accounts for the increased reorganization energy in azurin, and the increased hydrogen bonding at the copper site and/or conformational substates may explain the greater inhomogeneous component to the absorption line width in azurin.

Analysis of resonance Raman data on the blue copper site in pseudoazurin: Excited state π and σ charge transfer distortions and their relation to ground state reorganization energy

“…This method has been applied to BC proteins [56][57][58]. On a quantitative level, however, there are several issues in doing so.…”

Resonance Raman Intensities Demonstrate that C5 Substituents Affect the Initial Excited‐State Structural Dynamics of Uracil More than C6 Substituents

“…The addition of lithium sulfate did not noticeably change the absorption or resonance Raman spectra of 5,6‐ d 2 ‐U. The UV resonance Raman spectra of solutions containing 1 m m 5,6‐ d 2 ‐U and 0.3 m lithium sulfate internal standard were obtained at excitation wavelengths of 250, 257, 266, and 275 nm with laser powers of 5–12 mW, as previously described . Spectral slit widths were 5–7 cm −1 , and the total accumulation time was 20–30 min for each spectrum.…”

“…The methods used for converting the resonance Raman intensities into differential cross sections have been described previously . The experimental resonance Raman differential cross sections of sulfate used for the calculations were 3.30×10 −12 , 2.87×10 −12 , 2.42×10 −12 , and 2.05×10 −12 Å 2 molecule −1 sr −1 at λ =250, 257, 266, and 275 nm, respectively …”

The Role of Specific Amino Acid Residues in The Vibrational Properties of Plastocyanin

“…10,11,19 Measurement of the resonance Raman spectrum and determination of the intensities were repeated on three fresh samples of plastocyanin for each excitation wavelength. The methods used here for converting the resonance Raman intensities of plastocyanin into absolute cross sections 10 and for correcting for self-absorption effects 19 have been described previously. The cross sections of cacodylate used in the determination of absolute cross sections were calculated as described previously.…”