Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states

Abstract

Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the antiBrownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.photosynthesis | purple bacteria | fluorescence spectroscopy I n photosynthetic light harvesting, networks of antenna pigmentprotein complexes (PPCs) absorb sunlight, then efficiently transport the excitation through these networks to the reaction center, a PPC dedicated to charge separation (1, 2). Photosynthetic systems can complete this process both with near unity quantum efficiency and also function at light levels that provide photoenergy in excess of the capacity of downstream photochemistry. This is accomplished through mechanisms that dissipate harmful by-products produced by unused photoenergy (2, 3). The absorption, energy transport, and dissipation properties are governed by the balance of pigment-pigment and pigment-protein couplings (4). However, the molecular machinery responsible for this balance, and for the couplings themselves, is still poorly understood. This is because the couplings are highly sensitive to intermolecular distances. As a result of this sensitivity, the light-harvesting properties vary drastically among individual PPCs, because of small differences in protein conformation, and vary drastically even within a single PPC over time, because of protein fluctuations (2). In ensemble measurements, these drastic variations appear solely as a static, average value. Therefore, only through single-molecule spectroscopy can we investigate the photodynamics of individual PPCs, specifically how the absorption, energy transport, and dissipation of each change with time. We ...

Principles of light harvesting from single photosynthetic complexes

“…The dots form three clusters as determined by a Gaussian mixture model, shown with rainbow lines. These three clusters are labelled as states A, B and C. These results demonstrate that states A and B arise from a photoactivated, reversible quenching process [32]. As illustrated by the direct proportionality between states A and B, state B exhibits an increased rate of quenching, or nonradiative decay off the emissive state.…”

“…In contrast, the rate of transition from state B to state A is independent of excitation fluence, indicating a thermal process [32]. State C, however, most likely arises from an irreversible photodegradation process [32]. The two conformations of states A and B, and their dynamics allow a single protein to have both light-harvesting and photoprotective functionality.…”

“…The rate of transition from state A to state B increases roughly linearly with excitation fluence, indicating a photoactivated process [32]. Each photon deposits enough energy intensity (cpms) 12.5 into the protein to enable a conformational change, which causes the probability of the transition to increase with the number of photons [36].…”

Single-Molecule Identification of Quenched and Unquenched States of LHCII

“…The excited-state energies and dynamics are tuned by pigment-pigment and pigment-protein electrodynamic interactions, and so are exquisitely sensitive to changes in the molecular configuration of the complex 28 . As a result, conformational changes produce significantly altered photophysics [28][29][30][31][32][33] in Fig. 1) 43 , and we define these as "levels".…”

Real-time calibration of a feedback trap

“…7,8 Feedback traps can also be used to estimate the chemical properties of single molecules, including photodynamic and enzymatic properties of biomolecules 9 and the interplay between fluorescence spectroscopy and conformation at the single-molecule level. 10,11 The second kind of application has been to the study fundamental questions in statistical mechanics. The key feature of such traps is the ability to impose arbitrary, time-dependent virtual potentials on particles.…”