Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Abstract

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

The Unique Protein-To-Protein Carotenoid Transfer Mechanism

“…In turn, this indicates that dissociation of the otherwise very stable COCP dimer may be (i) the reason for the higher energy barrier and (ii) the cause for the initial delay (lag-phase) observed during carotenoid transfer from the wildtype COCP into Apo-OCP. Analogous s-shaped kinetics of OCP-related transitions were also observed during the fluorescence recovery after OCP-induced quenching of phycobilisomes in vivo, during which monomerization of FRP occurs and causes an increase in fluorescence recovery rate [14,37]. Considering the aforementioned facts, we assume that monomerization of COCP should occur first in order to initiate the transfer of the carotenoid molecule.…”

A Molecular Mechanism for Nonphotochemical Quenching in Cyanobacteria

“…The mutagenesis analysis shows that the amino acids (R60, W50 and D54)that form a network of hydrogen bonds between the two mFRP chains are essential for the enhancement of OCP r to OCP o conversion 21 . In addition, three previous studies suggest that FRP monomerizes when interacting with OCP analogs 14, 23, 53 . In this study, we saw a protein complex including mFRP, CTD and NTD after MS/MS dissociation (Fig.…”

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

“…We found that phosphate significantly reduces the amplitude of flash-induced spectral changes at 550 nm, accelerating the slow С3 component ( Table 1 ) and simultaneously reducing its yield ( Figure 1 ). This observation indicates that high concentrations of phosphate likely hinder domain separation of OCP, which is manifested in the rate of its back-conversion, and, therefore, accumulation of the physiologically active OCP R state, which explains why in vitro PBs quenching occurs only at a huge excess of OCP (~ 40 – 100 OCP per 1 PBs (16, 21, 23, 24)).…”

“…In order to test how the separation of domains affects the yields and lifetimes of different OCP intermediates with red-shifted absorption, we used the non-specific action of high concentrations of inorganic phosphate, a well-known kosmotrope, which is always present in buffers for experiments with PBs, thereby affecting the results of every in vitro fluorescence quenching experiment (16, 24). We found that phosphate significantly reduces the amplitude of flash-induced spectral changes at 550 nm, accelerating the slow С3 component ( Table 1 ) and simultaneously reducing its yield ( Figure 1 ).…”

“…Upon addition of FRP to OCP W288A we observed complex formation with 1 to 1 stoichiometry, and the absorption spectrum of this complex exhibited characteristic signatures of the orange state (referred to as “oranging”), presumably, due to OCP W288A stabilization by FRP (21). We hypothesized whether the FRP-induced stabilization of OCP W288A can be mimicked by kosmotropic phosphate anion, which acts non-specifically (19, 24, 40, 42), resulting in stabilization of CTD-NTD interactions (see Figure 1 and description in text). Increasing the phosphate concentration in OCP W288A solutions (pH was kept constant) caused a gradual increase of absorption in the blue spectral region and the appearance of vibronic substructure, a characteristic signature of the orange OCP form.…”