Multimeric and monomeric photosystem II supercomplexes represent structural adaptations to low- and high-light conditions.

An intriguing molecular architecture called the "semi-crystalline photosystem II (PSII) array" has been observed in the thylakoid membranes in vascular plants. It is an array of PSII-light-harvesting complex II (LHCII) supercomplexes that only appears in low light, but its functional role has not been clarified. Here, we identified PSII-LHCII supercomplexes in their monomeric and multimeric forms in low light-acclimated spinach leaves and prepared them using sucrose-density gradient ultracentrifugation in the presence of amphipol A8-35. When the leaves were acclimated to high light, only the monomeric forms were present, suggesting that the multimeric forms represent a structural adaptation to low light and that disaggregation of the PSII-LHCII supercomplex represents an adaptation to high light. Single-particle EM revealed that the multimeric PSII-LHCII supercomplexes are composed of two ("megacomplex") or three ("arraycomplex") units of PSII-LHCII supercomplexes, which likely constitute a fraction of the semi-crystalline PSII array. Further characterization with fluorescence analysis revealed that multimeric forms have a higher light-harvesting capability but a lower thermal dissipation capability than the monomeric form. These findings suggest that the configurational conversion of PSII-LHCII supercomplexes may serve as a structural basis for acclimation of plants to environmental light.

Ultrafast energy transfer between lipid-linked chromophores and plant light-harvesting complex II.

Light-Harvesting Complex II (LHCII) is a membrane protein found in plant chloroplasts that has the crucial role of absorbing solar energy and subsequently performing excitation energy transfer to the reaction centre subunits of Photosystem II. LHCII provides strong absorption of blue and red light, however, it has minimal absorption in the green spectral region where solar irradiance is maximal. In a recent proof-of-principle study, we enhanced the absorption in this spectral range by developing a biohybrid system where LHCII proteins together with lipid-linked Texas Red (TR) chromophores were assembled into lipid membrane vesicles. The utility of these systems was limited by significant LHCII quenching due to protein-protein interactions and heterogeneous lipid structures. Here, we organise TR and LHCII into a lipid nanodisc, which provides a homogeneous, well-controlled platform to study the interactions between TR molecules and single LHCII complexes. Fluorescence spectroscopy determined that TR-to-LHCII energy transfer has an efficiency of at least 60%, resulting in a 262% enhancement of LHCII fluorescence in the 525-625 nm range, two-fold greater than in the previous system. Ultrafast transient absorption spectroscopy revealed two time constants of 3.7 and 128 ps for TR-to-LHCII energy transfer. Structural modelling and theoretical calculations indicate that these timescales correspond to TR-lipids that are loosely- or tightly-associated with the protein, respectively, with estimated TR-to-LHCII separations of ∼3.5 nm and ∼1 nm. Overall, we demonstrate that a nanodisc-based biohybrid system provides an idealised platform to explore the photophysical interactions between extrinsic chromophores and membrane proteins with potential applications in understanding more complex natural or artificial photosynthetic systems.

Excitation quenching in chlorophyll-carotenoid antenna systems: 'coherent' or 'incoherent'.

Plants possess an essential ability to rapidly down-regulate light-harvesting in response to high light. This photoprotective process involves the formation of energy-quenching interactions between the chlorophyll and carotenoid pigments within the antenna of Photosystem II (PSII). The nature of these interactions is currently debated, with, among others, 'incoherent' or 'coherent' quenching models (or a combination of the two) suggested by a range of time-resolved spectroscopic measurements. In 'incoherent quenching', energy is transferred from a chlorophyll to a carotenoid and is dissipated due to the intrinsically short excitation lifetime of the latter. 'Coherent quenching' would arise from the quantum mechanical mixing of chlorophyll and carotenoid excited state properties, leading to a reduction in chlorophyll excitation lifetime. The key parameters are the energy gap, [Formula: see text] and the resonance coupling, J, between the two excited states. Coherent quenching will be the dominant process when [Formula: see text] i.e., when the two molecules are resonant, while the quenching will be largely incoherent when [Formula: see text] One would expect quenching to be energetically unfavorable for [Formula: see text] The actual dynamics of quenching lie somewhere between these limiting regimes and have non-trivial dependencies of both J and [Formula: see text] Using the Hierarchical Equation of Motion (HEOM) formalism we present a detailed theoretical examination of these excitation dynamics and their dependence on slow variations in J and [Formula: see text] We first consider an isolated chlorophyll-carotenoid dimer before embedding it within a PSII antenna sub-unit (LHCII). We show that neither energy transfer, nor the mixing of excited state lifetimes represent unique or necessary pathways for quenching and in fact discussing them as distinct quenching mechanisms is misleading. However, we do show that quenching cannot be switched 'on' and 'off' by fine tuning of [Formula: see text] around the resonance point, [Formula: see text] Due to the large reorganization energy of the carotenoid excited state, we find that the presence (or absence) of coherent interactions have almost no impact of the dynamics of quenching. Counter-intuitively significant quenching is present even when the carotenoid excited state lies above that of the chlorophyll. We also show that, above a rather small threshold value of [Formula: see text]quenching becomes less and less sensitive to J (since in the window [Formula: see text] the overall lifetime is independent of it). The requirement for quenching appear to be only that [Formula: see text] Although the coherent/incoherent character of the quenching can vary, the overall kinetics are likely robust with respect to fluctuations in J and [Formula: see text] This may be the basis for previous observations of NPQ with both coherent and incoherent features.

How carotenoid distortions may determine optical properties: lessons from the Orange Carotenoid Protein.

Carotenoids in photosynthetic proteins carry out the dual function of harvesting light and defending against photo-damage by quenching excess energy. The latter involves the low-lying, dark, excited state labelled S1. Here "dark" means optically-forbidden, a property that is often attributed to molecular symmetry, which leads to speculation that its optical properties may be strongly-perturbed by structural distortions. This has been both explicitly and implicitly proposed as an important feature of photo-protective energy quenching. Here we present a theoretical analysis of the relationship between structural distortions and S1 optical properties. We outline how S1 is dark not because of overall geometric symmetry but because of a topological symmetry related to bond length alternation in the conjugated backbone. Taking the carotenoid echinenone as an example and using a combination of molecular dynamics, quantum chemistry, and the theory of spectral lineshapes, we show that distortions that break this symmetry are extremely stiff. They are therefore absent in solution and only marginally present in even a very highly-distorted protein binding pocket such as in the Orange Carotenoid Protein (OCP). S1 remains resolutely optically-forbidden despite any breaking of bulk molecular symmetry by the protein environment. However, rotations of partially conjugated end-rings can result in fine tuning of the S1 transition density which may exert some influence on interactions with neighbouring chromophores.

A possible molecular basis for photoprotection in the minor antenna proteins of plants.

The bioenergetics of light-harvesting by photosynthetic antenna proteins in higher plants is well understood. However, investigation into the regulatory non-photochemical quenching (NPQ) mechanism, which dissipates excess energy in high light, has led to several conflicting models. It is generally accepted that the major photosystem II antenna protein, LHCII, is the site of NPQ, although the minor antenna complexes (CP24/26/29) are also proposed as alternative/additional NPQ sites. LHCII crystals were shown to exhibit the short excitation lifetime and several spectral signatures of the quenched state. Subsequent structure-based models showed that this quenching could be explained by slow energy trapping by the carotenoids, in line with one of the proposed models. Using Fluorescence Lifetime Imaging Microscopy (FLIM) we show that the crystal structure of CP29 corresponds to a strongly quenched conformation. Using a structure-based theoretical model we show that this quenching may be explained by the same slow, carotenoid-mediated quenching mechanism present in LHCII crystals.

The carotenoid pathway: what is important for excitation quenching in plant antenna complexes?

Plant light-harvesting is regulated by the Non-Photochemical Quenching (NPQ) mechanism involving the reversible formation of excitation quenching sites in the Photosystem II (PSII) antenna in response to high light. While the major antenna complex, LHCII, is known to be a site of NPQ, the precise mechanism of excitation quenching is not clearly understood. A preliminary model of the quenched crystal structure of LHCII implied that quenching arises from slow energy capture by Car pigments. It predicted a thoroughly quenched system but offered little insight into the defining aspects of this quenching. In this work, we present a thorough theoretical investigation of this quenching, addressing the factors defining the quenching pathway and possible mechanism for its (de)activation. We show that quenching in LHCII crystals is the result of slow energy transfer from chlorophyll to the centrally-bound lutein Cars, predominantly the Lut620 associated with the chlorophyll 'terminal emitter', one of the proposed in vivo pathways. We show that this quenching is rather independent of the particular species of Car and excitation 'site' energy. The defining parameter is the resonant coupling between the pigment co-factors. Lastly, we show that these interactions must be severely suppressed for a light-harvesting state to be recovered.

An 'all pigment' model of excitation quenching in LHCII.

The rapid, photoprotective down-regulation of plant light-harvesting in bright light proceeds via the non-photochemical quenching of chlorophyll excitation energy in the major photosystem II light-harvesting complex LHCII. However, there is currently no consensus regarding the precise mechanism by which excess energy is quenched. Current X-ray structures of this complex correspond to a dissipative conformation and therefore correct microscopic theoretical modelling should capture this property. Despite their accuracy in explaining the steady state spectroscopy of this complex, chlorophyll-only models (those that neglect the energetic role of carotenoids) do not explain the observed fluorescence quenching. To address this gap, we have used a combination of the semi-empirical MNDO-CAS-CI and the Transition Density Cube method to model all chlorophyll-carotenoid energy transfer pathways in the highly quenched LHCII X-ray structure. Our simulations reveal that the inclusion of carotenoids in this microscopic model results in profound excitation quenching, reducing the predicted excitation lifetime of the complex from 4 ns (chlorophyll-only) to 67 ps. The model indicates that energy dissipation proceeds via slow excitation transfer (>20 ps) from chlorophyll to the forbidden S1 excited state of the centrally bound lutein molecules followed by the rapid (∼10 ps) radiationless decay to the ground state, with the latter being assumed from experimental measurements of carotenoid excited state lifetimes. Violaxanthin and neoxanthin do not contribute to this quenching. This work presents the first all-pigment microscopic model of LHCII and the first attempt to capture the dissipative character of the known structure.

Photoprotective energy dissipation involves the reorganization of photosystem II light-harvesting complexes in the grana membranes of spinach chloroplasts.

Plants must regulate their use of absorbed light energy on a minute-by-minute basis to maximize the efficiency of photosynthesis and to protect photosystem II (PSII) reaction centers from photooxidative damage. The regulation of light harvesting involves the photoprotective dissipation of excess absorbed light energy in the light-harvesting antenna complexes (LHCs) as heat. Here, we report an investigation into the structural basis of light-harvesting regulation in intact spinach (Spinacia oleracea) chloroplasts using freeze-fracture electron microscopy, combined with laser confocal microscopy employing the fluorescence recovery after photobleaching technique. The results demonstrate that formation of the photoprotective state requires a structural reorganization of the photosynthetic membrane involving dissociation of LHCII from PSII and its aggregation. The structural changes are manifested by a reduced mobility of LHC antenna chlorophyll proteins. It is demonstrated that these changes occur rapidly and reversibly within 5 min of illumination and dark relaxation, are dependent on ΔpH, and are enhanced by the deepoxidation of violaxanthin to zeaxanthin.

Modeling the NMR signatures associated with the functional conformational switch in the major light-harvesting antenna of photosystem II in higher plants.

The major photosystem II antenna complex, LHCII, possesses an intrinsic conformational switch linked to the formation of a photoprotective, excitation-quenching state. Recent solid state NMR experiments revealed that aggregation-induced quenching in (13)C-enriched LHCII from C. reinhardtii is associated with changes to the chemical shifts of three specific (13)C atoms in the Chla conjugated macrocycle. We performed DFT-based NMR calculations on the strongly-quenched crystal structure of LHCII (taken from spinach). We demonstrate that specific Chla-xanthophyll interactions in the quenched structure lead to changes in the Chla(13)C chemical shifts that are qualitatively similar to those observed by solid state NMR. We propose that these NMR changes are due to modulations in Chla-xanthophyll associations that occur due to a quenching-associated functional conformation change in the lutein and neoxanthin domains of LHCII. The combination of solid-state NMR and theoretical modeling is therefore a powerful tool for assessing functional conformational switching in the photosystem II antenna.

Unravelling the fluorescence kinetics of light-harvesting proteins with simulated measurements.

The plant light-harvesting pigment-protein complex LHCII is the major antenna sub-unit of PSII and is generally (though not universally) accepted to play a role in photoprotective energy dissipation under high light conditions, a process known Non-Photochemical Quenching (NPQ). The underlying mechanisms of energy trapping and dissipation within LHCII are still debated. Various models have been proposed for the underlying molecular detail of NPQ, but they are often based on different interpretations of very similar transient absorption measurements of isolated complexes. Here we present a simulated measurement of the fluorescence decay kinetics of quenched LHCII aggregates to determine whether this relatively simple measurement can discriminate between different potential NPQ mechanisms. We simulate not just the underlying physics (excitation, energy migration, quenching and singlet-singlet annihilation) but also the signal detection and typical experimental data analysis. Comparing this to a selection of published fluorescence decay kinetics we find that: (1) Different proposed quenching mechanisms produce noticeably different fluorescence kinetics even at low (annihilation free) excitation density, though the degree of difference is dependent on pulse width. (2) Measured decay kinetics are consistent with most LHCII trimers becoming relatively slow excitation quenchers. A small sub-population of very fast quenchers produces kinetics which do not resemble any observed measurement. (3) It is necessary to consider at least two distinct quenching mechanisms in order to accurately reproduce experimental kinetics, supporting the idea that NPQ is not a simple binary switch.

The photoprotective molecular switch in the photosystem II antenna.

We have reviewed the current state of multidisciplinary knowledge of the photoprotective mechanism in the photosystem II antenna underlying non-photochemical chlorophyll fluorescence quenching (NPQ). The physiological need for photoprotection of photosystem II and the concept of feed-back control of excess light energy are described. The outline of the major component of nonphotochemical quenching, qE, is suggested to comprise four key elements: trigger (ΔpH), site (antenna), mechanics (antenna dynamics) and quencher(s). The current understanding of the identity and role of these qE components is presented. Existing opinions on the involvement of protons, different LHCII antenna complexes, the PsbS protein and different xanthophylls are reviewed. The evidence for LHCII aggregation and macrostructural reorganization of photosystem II and their role in qE are also discussed. The models describing the qE locus in LHCII complexes, the pigments involved and the evidence for structural dynamics within single monomeric antenna complexes are reviewed. We suggest how PsbS and xanthophylls may exert control over qE by controlling the affinity of LHCII complexes for protons with reference to the concepts of hydrophobicity, allostery and hysteresis. Finally, the physics of the proposed chlorophyll-chlorophyll and chlorophyll-xanthophyll mechanisms of energy quenching is explained and discussed. This article is part of a Special Issue entitled: Photosystem II.

Light-harvesting antenna composition controls the macrostructure and dynamics of thylakoid membranes in Arabidopsis.

We characterized a set of Arabidopsis mutants deficient in specific light-harvesting proteins, using freeze-fracture electron microscopy to probe the organization of complexes in the membrane and confocal fluorescence recovery after photobleaching to probe the dynamics of thylakoid membranes within intact chloroplasts. The same methods were used to characterize mutants lacking or over-expressing PsbS, a protein related to light-harvesting complexes that appears to play a role in regulation of photosynthetic light harvesting. We found that changes in the complement of light-harvesting complexes and PsbS have striking effects on the photosystem II macrostructure, and that these effects correlate with changes in the mobility of chlorophyll proteins within the thylakoid membrane. The mobility of chlorophyll proteins was found to correlate with the extent of photoprotective non-photochemical quenching, consistent with the idea that non-photochemical quenching involves extensive re-organization of complexes in the membrane. We suggest that a key feature of the physiological function of PsbS is to decrease the formation of ordered semi-crystalline arrays of photosystem II in the low-light state. Thus the presence of PsbS leads to an increase in the fluidity of the membrane, accelerating the re-organization of the photosystem II macrostructure that is necessary for induction of non-photochemical quenching.

Switching light harvesting complex II into photoprotective state involves the lumen-facing apoprotein loop.

In higher plants, high light conditions trigger the activation of non-photochemical quenching (NPQ), a process of photoprotective light energy dissipation, via acidification of the chloroplast lumen. Spectral changes occurring in the neoxanthin domain of the major light harvesting antenna complex (LHCII) have previously provided indirect evidence of a protein conformational switch during NPQ. We report here of two recombinant LHCII complexes mutated at the level of lumenal loop with altered quenching capacity with respect to the control. Replacement of the acidic lumenal-facing residue aspartate 111 (D111) with neutral valine (V111) yielded a recombinant complex with increased quenching capacity under low pH, due to a shift of the pK by 1 pH unit. The increase in total quenching was consistent with 40% reduction in the relative chlorophyll fluorescence lifetime and was accompanied by a lower energy emitting state of the mutant, as demonstrated by 77 K fluorescence spectroscopy. On the other hand, replacement of acidic glutamate 94 (E94) with glycine (G94) resulted in reduction of the fluorescence quenching yield attained at low pH. These results show for the first time that a subtle change in the LHCII apoprotein structure at the level of the lumenal loop induced by single aminoacid mutagenesis can affect protein sensitivity to pH leading to the establishment of NPQ. This work opens a potential avenue for manipulation of light harvesting efficiency in the natural antenna pigment-protein complexes that can be used for the creation of hybrid light energy conversion systems in future.

Origin of absorption changes associated with photoprotective energy dissipation in the absence of zeaxanthin.

To prevent photo-oxidative damage to the photosynthetic membrane in strong light, plants dissipate excess absorbed light energy as heat in a mechanism known as non-photochemical quenching (NPQ). NPQ is triggered by the trans-membrane proton gradient (ΔpH), which causes the protonation of the photosystem II light-harvesting antenna (LHCII) and the PsbS protein, as well as the de-epoxidation of the xanthophyll violaxanthin to zeaxanthin. The combination of these factors brings about formation of dissipative pigment interactions that quench the excess energy. The formation of NPQ is associated with certain absorption changes that have been suggested to reflect a conformational change in LHCII brought about by its protonation. The light-minus-dark recovery absorption difference spectrum is characterized by a series of positive and negative bands, the best known of which is ΔA(535). Light-minus-dark recovery resonance Raman difference spectra performed at the wavelength of the absorption change of interest allows identification of the pigment responsible from its unique vibrational signature. Using this technique, the origin of ΔA(535) was previously shown to be a subpopulation of red-shifted zeaxanthin molecules. In the absence of zeaxanthin (and antheraxanthin), a proportion of NPQ remains, and the ΔA(535) change is blue-shifted to 525 nm (ΔA(525)). Using resonance Raman spectroscopy, it is shown that the ΔA(525) absorption change in Arabidopsis leaves lacking zeaxanthin belongs to a red-shifted subpopulation of violaxanthin molecules formed during NPQ. The presence of the same ΔA(535) and ΔA(525) Raman signatures in vitro in aggregated LHCII, containing zeaxanthin and violaxanthin, respectively, leads to a new proposal for the origin of the xanthophyll red shifts associated with NPQ.

Electronic spectra of structurally deformed lutein.

Quantum chemical calculations have been employed for the investigation of the lowest excited electronic states of lutein, with particular reference to its function within light harvesting antenna complexes of higher plants. Through comparative analysis obtained by using different methods based on gas-phase calculations of the spectra, it was determined that variations in the lengths of the long C-C valence bonds and the dihedral angles of the polyene chain are the dominant factors in determining the spectral properties of Lut 1 and Lut 2 corresponding to the deformed lutein molecules taken from crystallographic data of the major pigment-protein complex of photosystem II. By MNDO-CAS-CI method, it was determined that the two singlet B(u) states of lutein (nominally 1B(u)(-)* and 1B(u)(+)) arise as a result of mixing of the canonical 1B(u)(-) and 1B(u)(+) states of the all-trans polyene due to the presence of the ending rings in lutein. The 1B(u)(-)* state of lutein is optically allowed, while the 1B(u)(-) of a pure all-trans polyene chain is optically forbidden. As demonstrated, the B(u) states are much more sensitive to minor distortions of the conjugated chain due to mixing of the canonical states, resulting in states of poorly defined particle-hole symmetry. Conversely, the A(g) states are relatively robust with respect to geometric distortion, and their respective inversion and particle-hole symmetries remain relatively well-defined.

Understanding Carotenoid Dynamics via the Vibronic Energy Relaxation Approach.

Carotenoids are an integral part of natural photosynthetic complexes, with tasks ranging from light harvesting to photoprotection. Their underlying energy deactivation network of optically dark and bright excited states is extremely efficient: after excitation of light with up to 2.5 eV of photon energy, the system relaxes back to ground state on a time scale of a few picoseconds. In this article, we summarize how a model based on the vibrational energy relaxation approach (VERA) explains the main characteristics of relaxation dynamics after one-photon excitation with special emphasis on the so-called S* state. Lineshapes after two-photon excitation are beyond the current model of VERA. We outline this future line of research in our article. In terms of experimental method development, we discuss which techniques are needed to better describe energy dissipation effects in carotenoids and within the first solvation shell.

Trivial Excitation Energy Transfer to Carotenoids Is an Unlikely Mechanism for Non-photochemical Quenching in LHCII.

Higher plants defend themselves from bursts of intense light via the mechanism of Non-Photochemical Quenching (NPQ). It involves the Photosystem II (PSII) antenna protein (LHCII) adopting a conformation that favors excitation quenching. In recent years several structural models have suggested that quenching proceeds via energy transfer to the optically forbidden and short-lived S 1 states of a carotenoid. It was proposed that this pathway was controlled by subtle changes in the relative orientation of a small number of pigments. However, quantum chemical calculations of S 1 properties are not trivial and therefore its energy, oscillator strength and lifetime are treated as rather loose parameters. Moreover, the models were based either on a single LHCII crystal structure or Molecular Dynamics (MD) trajectories about a single minimum. Here we try and address these limitations by parameterizing the vibronic structure and relaxation dynamics of lutein in terms of observable quantities, namely its linear absorption (LA), transient absorption (TA) and two-photon excitation (TPE) spectra. We also analyze a number of minima taken from an exhaustive meta-dynamical search of the LHCII free energy surface. We show that trivial, Coulomb-mediated energy transfer to S 1 is an unlikely quenching mechanism, with pigment movements insufficiently pronounced to switch the system between quenched and unquenched states. Modulation of S 1 energy level as a quenching switch is similarly unlikely. Moreover, the quenching predicted by previous models is possibly an artifact of quantum chemical over-estimation of S 1 oscillator strength and the real mechanism likely involves short-range interaction and/or non-trivial inter-molecular states.

A Protein Environment-Modulated Energy Dissipation Channel in LHCII Antenna Complex.

The major light-harvesting complex of photosystem II (LHCII) is the main contributor to sunlight energy harvesting in plants. The flexible design of LHCII underlies a photoprotective mechanism whereby this complex switches to a dissipative state in response to high light stress, allowing the rapid dissipation of excess excitation energy (non-photochemical quenching, NPQ). In this work, we locked single LHCII trimers in a quenched conformation after immobilization of the complexes in polyacrylamide gels to impede protein interactions. A comparison of their pigment excited-state dynamics with quenched LHCII aggregates in buffer revealed the presence of a new spectral band at 515 nm arising after chlorophyll excitation. This is suggested to be the signature of a carotenoid excited state, linked to the quenching of chlorophyll singlet excited states. Our data highlight the marked sensitivity of pigment excited-state dynamics in LHCII to structural changes induced by the environment.

Structural Basis for Allosteric Regulation in the Major Antenna Trimer of Photosystem II.

The allosteric regulation of protein function proves important in many life-sustaining processes. In plant photosynthesis, LHCII, the major antenna complex of Photosystem II, employs a delicate switch between light harvesting and photoprotective modes. The switch is triggered by an enlarged pH gradient (ΔpH) across the thylakoid membranes. Using molecular simulations and quantum calculations, we show that ΔpH can tune the light-harvesting potential of the antenna via allosteric regulation of the excitonic coupling in chlorophyll-carotenoid pairs. To this end, we propose how the LHCII excited state lifetime is coupled to the environmental conditions. In line with experimental findings, our theoretical model provides crucial evidence toward the elucidation of the photoprotective switch of higher plants at an all-atom resolution.

The full dynamics of energy relaxation in large organic molecules: from photo-excitation to solvent heating.

In some molecular systems, such as nucleobases, polyenes or the active ingredients of sunscreens, substantial amounts of photo-excitation energy are dissipated on a sub-picosecond time scale, raising questions such as: where does this energy go or among which degrees of freedom it is being distributed at such early times? Here we use transient absorption spectroscopy to track excitation energy dispersing from the optically accessible vibronic subsystem into the remaining vibrational subsystem of the solute and solvent. Monitoring the flow of energy during vibrational redistribution enables quantification of local molecular heating. Subsequent heat dissipation away from the solute molecule is characterized by classical thermodynamics and molecular dynamics simulations. Hence, we present a holistic approach that tracks the internal temperature and vibronic distribution from the act of photo-excitation to the restoration of the global equilibrium. Within this framework internal vibrational redistribution and vibrational cooling are emergent phenomena. We demonstrate the validity of the framework by examining a highly controversial example, carotenoids. We show that correctly accounting for the local temperature unambiguously explains their energetically and temporally congested spectral dynamics without the ad hoc postulation of additional 'dark' states. An immediate further application of this approach would be to monitor the excitation and thermal dynamics of pigment-protein systems.