Rafael Picorel

Langmuir-Blodgett and X-ray Diffraction Studies of Isolated Photosystem II Reaction Centers in Monolayers and Multilayers: Physical Dimensions of the Complex*

Abstract— The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water‐oxidation process of photosynthesis. Unlike the bacterial RC complex, an X‐ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross‐sectional area of an isolated RC in a condensed monolayer film. Low‐angle X‐ray diffraction results obtained by examining Langmuir‐Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z‐direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical‐type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. PL Physiol. 22,161–166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron‐density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.

SURFACE‐ENHANCED RESONANCE RAMAN SCATTERING SPECTROSCOPY AS A SURFACE TOPOGRAPHY PROBE IN PLANT PHOTOSYNTHETIC MEMBRANES

Strong resonance Raman (RR) and surface‐enhanced resonance Raman scattering (SERRS) signals from carotenoids were detected from thylakoid (stromal‐side out) vesicles and inside‐out (lumenal‐side out) vesicles isolated from spinach chloroplasts. The intensity of the signals from both types of membranes was comparable, indicating that plant carotenoids are exposed on or close to both surfaces or sides of the thylakoid membrane. This is in contrast to previous studies with bacterial photosynthetic membranes (Picorel et al., 1988, J. Biol. Chem. 263, 4374–4380; and 1990, Biochemistry29, 707–712) that show carotenoids selectively located on the cytoplasmic side. In addition; strong RR and SERRS signals were detected from stacked and unstacked photosystem‐II‐enriched membrane fragments, demonstrating that carotenoids are also exposed on both surfaces of the appressed region of the thylakoid membrane. Antibodies against the photosystem (PS) II extrinsic proteins blocked SERRS signals from stacked PS II membrane fragments, but only partially affected the SERRS signals from unstacked membranes. The results indicate that these antibodies, which preferentially cover the surface of the original lumenalside of the appressed region, act as spacers between the membrane and SERRS electrode surfaces. The original stromal‐side of the appressed region is unaffected. These findings verify the distance sensitivity of the SERRS technique and underscore the above conclusion about the location of carotenoids in the appressed regions. Finally, SERRS signals are sensitive to membrane aging and storage temperature; caution is suggested to those applying SERRS spectroscopy to intact membrane systems.

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions.

Abstract Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment–pigment and pigment–protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment–protein complexes from different sources or in complexes altered chemically or genetically.

STRUCTURAL AND FUNCTIONAL INTEGRITY OF THE PHOTOSYSTEM II REACTION CENTER ON SILVER ELECTRODES: FLUORESCENCE AND REDOX PROBES

Two simple and sensitive methods have been developed to assess the structural and functional integrity of isolated photosystem II reaction centers deposited on a roughened Ag electrode. Surface‐enhanced resonance Raman scattering (SERRS) spectra useful for ascertaining structural information can be obtained from biological materials with this technique. The first method presented is based on observing differences in the fluorescence emission properties of reaction centers; these depend on the activity of the material. The second is based on the observation of changes in Raman bands that are sensitive to the redox state of cytochrome b559 present in the reaction center complex. It is concluded that the conditions used here to obtain SERRS spectra do not affect the structural or functional integrity of the isolated photosystem II reaction center complex. In principle these approaches also could be used with other chromoproteins.