Bernard Rospendowski

Surface enhanced resonance Raman scattering (SERRS) as a probe of the structural differences between the Pr and Pfr forms of phytochrome

Surface enhanced resonance Raman scattering (SERRS) spectra have been obtained from the active, far‐red light absorbing (Pfr) and biologically inactive (Pr) forms of phytochrome adsorbed on silver colloids. Substantial differences between the SERRS spectra of the two forms in the low and high wavenumber regions are observed using 406.7 nm wavelength excitation. These differences reinforce those seen with 413.1 nm wavelength excitation in the high wavenumber region. Simultaneously, extensive differences are observed in the SERRS obtained from the same form in the low wavenumber region using 406.7 nm, as compared with 413.1 nm wavelength excitation. The relative intensity differences observed for the two forms, and those obtained using two slightly different excitation wavelengths to illuminate the same form, suggest that some type of subtle, proteincontrolled structural variation is responsible for the spectroscopic differences. A Z → E isomerization during the Pr → Pfr phototransformation is consistent with the SERRS data, although the overall chromophore conformations are most likely conserved for the native Pr‐ and Pfr‐phytochrome species. Slight out‐of‐plane ring twisting, accompanying the Pr → Pfr photoisomerization, may be responsible for the large difference in the spectroscopic properties of the native Pr and Pfr chromophores.

Overtone and combination band Raman spectra of α-copper phthalocyanine

Compacted electrodes made by pressing α-copper phthalocyanine (αCuPc) and silver metal powder provide an effective method to measure resonance Raman scattering from αCuPc. We report first and very weak second overtone and combination bands of αCuPc. They are based on progressions in ν3, ν4, ν7 and ν15 modes. Three of these are A1g vibrations of αCuPc, indicating a much stronger symmetric contribution to overtone scattering than is the case in porphyrins. Overtone profiles at 0 V vs. SCE arise mainly from scattering from 0 to 1 vibronic overtone states with evidence of higher state contributions in some cases. This is in contrast to the more varied profiles of fundamental modes and is as expected from theory. The results support the view that Raman scattering from αCuPc involves a Jahn–Teller distorted excited state and is due to A as well as B term enhancement.

Surface-Enhanced Resonance Raman Scattering from Cytochromes Cand P-450 on Bare and Phospholipid-Coated Silver Substrates

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) have demonstrated that the native integrity of this heme-containing protein is preserved in the presence of citrate-reduced sols. Cyt c SERRS spectra exhibit similar spin and oxidation markers as the resonance Raman scattering (RRS) signals displayed by the native protein solution. Previous SERRS investigations of cyt c showed that the protein denatures at the substrate surface. This hemoprotein denaturation was attributed to an electrostatic interaction between the porphyrin and the SERRS-active substrate. The results presented in this study suggest that the citrate ions prevent the biomolecule from directly interacting with the silver-sol surface. The native state of cyt c is also retained with the addition of phosphatidylcholine and/or phosphate buffer. Although the SERRS bandshifts correlate with the RRS spectra, the relative intensities of specific cyt c frequencies change in the presence of adsorbed citrate, phosphate buffer, and phosphatidylcholine. These compounds may influence the orientation of the protein at the substrate surface, which would account for the spectral intensity differences. In order to study the orientation effects further, additional studies of cyt c adsorbed onto silver electrodes and thin silver films were conducted. A comparison of the SERRS relative band intensities and frequency bandshifts shows that the spectra of the protein adsorbed onto phospholipid-coated substrates closely correspond to the RRS cyt c solution spectra. In contrast to the behavior exhibited by cyt c, cytochrome P-450 (cyt P-450) adsorbed onto a phosphatidylcholine silver-sol substrate leads to a low to high spin-state conversion. The shifts in the spin-state markers are ascribed to a strong interaction of the cyt P-450 with the phospholipid coating.