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

Abstract

The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCP^O state to the red active signaling state, OCP^R, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCP^O suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCP^R. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCP^R to OCP^O. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.

Figure 1

(A) flash-induced transitions of OCP absorption at 550 nm approximated by multiexponential decay: OCP – red, OCP in 1 M phosphate – grey; OCP in the presence of FRP (1/1.6 concentration ratio) – blue; ΔNTE OCP in the presence of FRP (1/1.6 concentration ratio) – black. Note the logarithmic timescale covering almost 7 orders of magnitude. (B) dependency of the photoconversion amplitude on the energy of the laser flash for OCP in the presence of FRP (1/1.6 concentration ratio). (C) Arrhenius plot of the fast (C1) and the slow (C3) components’ rate constants of absorption changes of OCP in the presence of FRP. Experiments were conducted in the range of temperatures from 6 to 37 °C.

Figure 2

Changes of intrinsic Trp fluorescence of OCP associated with OCP^R-OCP^O conversion. (A) a typical set of 400 fluorescence decay kinetics normalized to maximum intensity measured at 10 °C successively after switching off the actinic light, causing conversion of OCP from the red to the orange form. Black lines indicate the levels of fluorescence intensity. (B) time courses of fluorescence intensity changes at 350 nm upon the OCP^R-OCP^O transition at temperatures indicated by different colors as described in the inset (values in °C). Each experimental point was obtained by integration of the corresponding Trp fluorescence decay curve. Note the logarithmic scales of both axes. (C) normalized Trp fluorescence decay kinetics of the photoactivated (OCP^R) and back-converted (OCP^O, after 60 minutes in the dark) protein. The experiment was conducted at 15 °C. (D) Arrhenius plots for the OCP^R-OCP^O relaxation measured as absorption changes at 550 nm (Abs, blue circles), changes of Trp fluorescence intensity (Trp fl, red squares) and changes of average Trp fluorescence lifetime (EET, open circles) calculated from the data presented in (B). (E) characteristic time courses of OCP^R-OCP^O transitions of OCP measured as changes of carotenoprotein absorption (blue) at 550 nm, and Trp fluorescence intensity (red circles) and lifetime (or EET, open circles) at 350 nm. Transitions were measured at 15 °C, the concentration of the sample was identical for fluorescence and absorption measurements. The values of O.D., fluorescence intensity and lifetimes were normalized to unity. The time-course of the static quenching (red squares) was calculated from the changes of overall fluorescence intensity $\frac{Fl(t)}{{Fl}_R}$ and average lifetimes $\frac{\tau (t)}{{\tau }_R}$ as $\frac{Q(t)}{Q_R}=1-\frac{\tau (t)}{{\tau }_R}+\frac{Fl(t)}{{Fl}_R}$ .

Figure 3

Effects of phosphate on OCP^W288A. (A) absorption spectrum of OCP^W288A in 0.8 M phosphate buffer. The spectrum represents a mixture of orange and violet forms. The dotted line shows the absorption spectrum of the OCP^Y201A/W288A mutant, which is identical to the initially purple OCP^W288A, but not sensitive to phosphate. The inset shows images of cuvettes containing solutions of OCP^W288A in buffers with different phosphate concentration (indicated) . (B) photocycle of OCP^W288A measured as changes of O.D. at 550 nm upon illumination by actinic light. (C) Raman spectra of OCP^W288A without and with phosphate. (D) photoactivity of wild-type OCP and the OCP^W288A mutant at different phosphate concentrations. Photoactivity was determined as changes of O.D. at 550 nm normalized to the carotenoid concentration in the sample. Maximum changes of O.D. at 550 nm for wild-type OCP in the absence of phosphate were set to 100%.

Figure 4

Quenching of Trp fluorescence in OCP and COCP by the carotenoid cofactor and iodide. (A) location of all Trp residues and Tyr-201 in the crystal structure of OCP in the orange state (PDB:3MG1). The carotenoid is shown by orange spheres, Trp residues and Tyr-201 are shown as violet and pink sticks, respectively. (B) fluorescence decay kinetics of COCP (violet) and Apo-COCP (black) measured at 25 °C. The protein concentrations were equal in both samples. (C,D) quenching of OCP^O and OCP^R Trp fluorescence by increasing concentrations of iodide. At each iodide concentration, picosecond fluorescence decays were measured. All experiments with iodide were carried out at 2 °C in order to reduce the rate of OCP^R to OCP^O back-conversion. (E) dependencies of quenching efficiency on iodide concentration calculated as changes of fluorescence intensities and average lifetimes from the data presented in panels C and D. (F) modified Stern-Volmer plots of Trp fluorescence quenching by iodide approximated by linear functions. The ordinate intercepts define the reciprocal values of the fraction of accessible Trp residues (f _a). The protein concentrations were equal in all samples.

Figure 5

Novel aspects of the OCP photocycle. See text for details.