Fluorescence Polarization Studies of B-Phycoerythrin Oriented in Polymer Film6

Photochemistry and Photobiology, Jan 2004 by Frackowiak, Danuta, Ptak, Arkadiusz, Gryczynski, Zygmunt, Gryczynski, Ignacy, Et al

ABSTRACT

Polarized steady-state fluorescence and fluorescence excitation spectra as well as time-resolved fluorescence for B-phycoerythrin (B-PE) from red algae, Porphyridium cruentum, embedded in polyvinyl stretched films were measured. The lifetimes of polarized fluorescence were analyzed using exponential components and fractal models. The interactions between various chromophores of the pigment-protein complexes investigated were discussed. The anisotropy of fluorescence excitation spectra differs from the anisotropy of absorption spectra and depends on the wavelength of observation. This shows that differently oriented chromophores take part in various paths of excitation energy transfer (ET) or change their excitation into heat with various efficiencies (or both). Also, analysis of time-resolved fluorescence measured in various spectral regions gives different polarized components of emission. Fractal analysis of lifetimes, done under supposition of the Foerster resonance ET mechanism, suggests different arrangements of energy donors and acceptors for molecules absorbing in different spectral regions. It shows that several fractions of differently oriented "forms" of chromophores exhibiting different spectral properties occur in B-PE complexes. Small changes in the orientation of the chromophores can be followed by modification of the path of excitation energy migration. Based on the results obtained a new reorientational mechanism of the State 1 [arrow right] State 2 transition was proposed: Even small conformational modifications of biliproteins, which could be caused in vivo by the change in the conditions of preillumination of bacteria, are able to modify the path of excitation ET. Such a reorientation may be responsible for the change in the partition of biliprotein excitation energy between photosystem II (PSII) and PSI (State 1 [arrow right] State 2 transition). The proposed mechanism needs further verification by the investigation of whole bacteria cells.

Abbreviations: B-PE, B-phycoerythrin; CD, circular dichroism; ET, energy transfer; FRET, Foerster resonance energy transfer; LD, linear dichroism; PBS, phycobilisome; PE, phycoerythrin; PEB, phycoerythrobilin; PSII, photosystem II; PUB, phycourobilin; PVA, polyvinyl alcohol; TM, transition moment.

INTRODUCTION

Biliproteins are light-harvesting pigment-protein complexes that absorb light in the region of low absorption of chlorophylls (1-3). Phycobilisomes (PBS)-the giant complexes containing biliproteins-are the main light harvester in cyanobacteria and in red algae. A major part of PBS excitation is transferred to photosystem II (PSII), whereas a minor part of excitation is transferred to PSI (1,2). Fractions of excitation energy transferred to PSII and to PSI depend on the "light history" and osmolality of cell suspension (4). In hypoosmotic suspension of the light acclimated cells (in State 1) PBS transfer most of their excitation energy to PSII, whereas some increase in the excitation transfer to PSI is observed as a result of dark adaptation of cells (in State 2) (5,6). This transition in states (State 1 [arrow right] State 2) can be explained based on one of the following three models: (1) a strong coupling between PBS and PSII in State 1-the change in this coupling is responsible for the transition to State 2; (2) a "spillover" model, in which energy is transferred from PSII directly to PSI; and (3) a "mobile PBS" model that supposes detachment of PBS from PSII and its movement to PSI. There are several experimental data supporting each of these models (4,6-9).

The efficiency of excitation energy transfer (ET) between antenna complexes depends not only on the overlapping of their absorption and emission spectra but also on the mutual orientations of their chromophores. These orientations can be investigated by polarized light absorption and fluorescence spectroscopy (10-12). Previously, we investigated possible paths of the excitation ET between various antenna complexes of cyanobacteria and red algae in whole cells (13) and in whole PBS (14-16) oriented in an anisotropic polymer matrix. Previous results concerning whole PBS obtained from organisms adapted to green or red radiation (17) show that light adaptation changes the ET from phycoerythrin (PE) to allophycocyanin and also has influence on the orientation of pigment-protein complexes (18,19). The paths of the excitation ET in cyanobacteria and in red algae are often investigated on various systems constructed of biliprotein complexes (6,20,21). The orientation of chromophores inside monomers built from subunits [alpha] and [beta] and inside dimers ([alpha][beta])2 of various biliproteins have been intensively investigated by MacColl et al. (10-12), and models of chromophore orientations have been proposed. These investigations were carried out for several biliprotein suspensions using a polarized light photoselection effect.

In the present work we have investigated the excitation ET inside a more complicated system, that is, in the whole B-phycoerythrin (B-PE) containing six [alpha], six [beta] and one [gamma] subunits ([alpha]^sub 6^[beta]^sub 6^[gamma]^sub 1^) obtained from red algae, Porphyridium cruentum. Such type of biliprotein complexes have not been yet investigated. In our experiments the B-PE complexes are embedded in rigid polyvinyl alcohol (PVA) films. Our previous investigations (13-16) show that results obtained for cells or for complexes embedded in PVA film can be useful in the interpretation of processes occurring in vivo. Some of the samples were oriented by PVA film stretching. B-PE contains two types of chromophores: phycoerythrobilin (PEB) (absorption in the 545 and 565 nm regions) and phycourobilin (PUB) (absorption about 495 nm), located in [alpha], [beta], and [gamma] subunits (1,2). Various types of [gamma] subunits were found, but B-PE was always built from the same combination of subunits: [alpha]^sub 6^[beta]^sub 6^[gamma]^sub 1^ (1-2). Two PEB chromophores are attached to subunit ? and three PEB to [beta], whereas usually two PEB and two PUB chromophores are attached to the [gamma] subunit (1,2). PE from various organisms exhibit different spectral properties (1), which suggests their different structure and the various interactions between chromophores. To establish the type of interactions between chromophores in B-PE, polarized absorption, fluorescence and fluorescence excitation as well as the lifetimes of various polarized components of fluorescence were measured and analyzed.