Fluorescence Micro-Spectroscopy Study of Individual Photosynthetic Membrane Vesicles and Light-Harvesting Complexes

Abstract

Single-molecule spectroscopy, by getting rid of unwanted ensemble averaging effects, has proved to be a very valuable tool in the research of individual photosynthetic light-harvesting (LH) complexes. However, to learn about real photosynthetic processes the minimal unit to study is a single photosynthetic membrane complete with all elements of its machinery. In the present work, the fluorescence spectra of excitons in lone intracytoplasmic (IC) photosynthetic membrane vesicles of the wild type purple bacterium Rhodobacter sphaeroides that involve peripheral (LH2) and core (RC-LH1-PufX) antenna pigment-protein complexes were investigated at ambient temperature under continuous-wave laser excitation into the Q(x) absorption band of the bacteriochlorophyll-a (BChl) chromophores at 594 nm. In parallel, the spectra of mutant membrane vesicles occupied by just one type of complexes (either LH2 or RC-LH1-PufX) and the spectra of individual purified LH2 and RC-LH1-PufX complexes were measured. The fluorescence from full IC membranes shows a high sensitivity to excitation intensity, being varied over more than four orders of magnitude between 0.1 W/cm(2) and 2 kW/cm(2). At low to moderate excitation intensities, the spectra of IC membranes could be well reproduced by its component spectra, the ratio of the spectra related to peripheral and core complexes being the only adjustable parameter. The spectra of both intact chromatophores and individual membrane components recorded over 1-50 s experimental time frames are robust, strongly suggesting that large spectral fluctuations hardly play a role in the functional photosynthetic process. The significant, up to 14 times, variation of the LH2 and LH1 emission ratio observed in individual IC membranes could be related to variations in the stoichiometric ratio of the peripheral and core complexes. Evidence was found for the presence of LH2 parts that are detached from efficient energy transfer pathways. Upon strong and prolonged illumination, the membrane spectra reveal significant permanent modifications. These alterations, which mostly concern peripheral antenna complexes, were shown to be due to photo-oxidation of various numbers of BChl molecules in the B850 compartment of LH2.

High temperature limit of photosynthetic excitons

“…By spectral position and shape, this thermo-product band can be assigned to individual solubilized BChl a molecules, irreversibly detached from the binding pockets of LH2 due to denaturation of the protein. The weak shoulders observable at 70 °C are likely associated with the forms of LH2 where the BChl a pigments are either partially (peak around 845 nm) or fully oxidized (695 nm) 19 , 20 . It is important to notice that at all temperatures the shape of the residual absorption spectrum (shown in red in the inset of Fig.…”

Vibronic Origin of the Qy Absorption Tail of Bacteriochlorophyll a Verified by Fluorescence Excitation Spectroscopy and Quantum Chemical Simulations

“…17 The solvent was dried by storing on molecular sieves (3 Å 1/16 by Wako) for at least 24 h prior to use. The measurements with spectral resolution of 1.5 nm were carried out at an ambient temperature of 22 ± 1 °C using a home-built microspectroscopy system 18 composed of an Olympus IX-71 inverted microscope furnished with a Mitutoyo 10× long-working-distance objective (NA = 0.28) and an Andor Shamrock 303i spectrometer equipped with an Andor spectroscopic camera iDus 420. The absorption spectrum was measured applying a standard tungsten halogen illumination lamp.…”

Establishment of the Qy Absorption Spectrum of Chlorophyll a Extending to Near-Infrared

“…The measurements with spectral resolution of 1.5 nm were carried out using a home-built microspectroscopy system [ 42 ] composed of an Olympus IX-71 inverted microscope equipped with a 10x long-working-distance objective (NA = 0.28) (Mitutoyo, Kawasaki, Japan) and a Shamrock 303i spectrometer (Andor, Belfast, UK) together with an Andor spectroscopic camera iDus 420. Absorption/absorptance spectra were measured applying a standard tungsten halogen illumination lamp.…”