Regulation of Light Harvesting in the Green Alga Chlamydomonas reinhardtii: The C‑Terminus of LHCSR Is the Knob of a Dimmer Switch Nicoletta Liguori, † Laura M. Roy, † Milena Opacic, † Gre ́ gory Durand, ‡,# and Roberta Croce* ,† † Department of Physics and Astronomy and Institute for Lasers, Life and Biophotonics, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands ‡ Equipe Chimie Bioorganique et Syste ̀ mes Amphiphiles, Universite ́ d’Avignon, 33 rue Louis Pasteur, F-84000 Avignon, France # Institut des Biomole ́ cules Max Mousseron (UMR 5247), 15 avenue Charles Flahault, F-34093 Montpellier Cedex 05, France * S Supporting Information ABSTRACT: Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light- harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environ- ment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR’s uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex. T he capture and storage of light energy by photosynthetic organisms is the process that sustains virtually all life on earth, but it is also a hazardous business. If the absorbed energy exceeds the capacity of the metabolic reactions, it can result in photo-oxidation events that can ultimately result in the organism’s death. 1 Plants and algae have evolved elaborate mechanisms to protect themselves against oxidative damage. 1,2 Collectively known as non-photochemical quenching (NPQ), these multicomponent mechanisms serve to dissipate excess absorbed energy as heat. It is known that this process is triggered by low luminal pH, 1 an indication that the electron transport chain in the photosynthetic apparatus is under stress, but the exact action mechanism is a matter of debate. Members of a subfamily of light-harvesting complex (LHC) genes are known to be major players in this process. 2 While the PsbS protein required for qE, the fast component of NPQ, is constitutively expressed in higher plants and does not bind pigments, 1 algae and mosses 3 require the expression of a stress- related pigment-binding complex previously indicated as LI818. 4,5 LHCSR, as it is now known, has recently been identified as the key component to activate qE in the model organism Chlamydomonas reinhardtii. 3 LHCBs, the light-harvesting antennae of photosystem II (PSII), were also suggested to have a role in the quenching process as sites of chlorophyll energy dissipation. 6,7 Currently, the hypothesis that PsbS and LHCSR represent active triggers of a conformational switch after sensing lumen acidification is the most accepted one. 8,9 This switch is in turn hypothesized to initiate a functional rearrangement of the whole PSII, including conformational changes in LHCb antennae, leading to energy dissipation. 10−12 However, the nature of the quencher still remains a matter of debate, 6,13−15 and the fact that LHCSR binds pigments, while PsbS does not, suggests different quenching mechanisms in plants and algae. Nevertheless, in all organisms, the necessary condition to induce structural interconversion is the availability of pH sensors. PsbS has been shown to possess two lumen-exposed acidic residues which are necessary for its function in qE in plants. 16 Although the availability of one or two pH-sensitive residues was also reported for nearly every LHCb, 17−19 a self-assisted conforma- tional switch to a dissipative state upon lumen acidification for the single PSII antenna has not been clearly demonstrated. Indeed, most of the studies showing pH-dependent quenching of LHCs 19−22 have been performed upon detergent removal, thus inducing aggregation. Oligomerization is well known to cause high degrees of quenching, 23,24 with the dissipation magnitude depending on the size of the aggregates. 24,25 This makes it impossible to discriminate between the direct effect of the pH on observed quenching and that of aggregation, and it is then easy to understand the primary importance of elucidating the direct effect of pH on the induction of energy dissipation. In this work, we employed a new methodology to investigate in vitro the response and sensitivity to pH variations of two different systems. First, we studied the main LHC complex, LHCII, in both trimeric and monomeric forms, aiming to characterize its sensitivity to its environment. Next we focused on the pH response of the stress-related LHCSR from C. reinhardtii, with the aim of understanding its action mechanism in triggering qE activation. 3 Finally, we investigated possible bridges between its optical properties and structural features by mutating all protonable residues in its C-terminus. To obtain reliable data, it is essential to be able to perform the experiments at different pH values without incurring aggregation or misfolding of the complexes. Indeed, aggrega- tion is not the only undesired side effect deriving from an acid Received: October 21, 2013 Published: November 21, 2013 Communication pubs.acs.org/JACS © 2013 American Chemical Society 18339 dx.doi.org/10.1021/ja4107463 | J. Am. Chem. Soc. 2013, 135, 18339−18342