Infrared Signatures of Phycobilins within the Phycocyanin 645 Complex

Abstract

Aquatic photosynthetic organisms evolved to use a variety of light frequencies to perform photosynthesis. Phycobiliprotein phycocyanin 645 (PC645) is a light-harvesting complex in cryptophyte algae able to transfer the absorbed green solar light to other antennas with over 99% efficiency. The infrared signatures of the phycobilin pigments embedded in PC645 are difficult to access and could provide useful information to understand the mechanism behind the high efficiency of energy transfer in PC645. We use visible-pump IR-probe and two-dimensional electronic vibrational spectroscopy to study the dynamical evolution and assign the fingerprint mid-infrared signatures to each pigment in PC645. Here, we report the pigment-specific vibrational markers that enable us to track the spatial flow of excitation energy between the phycobilin pigment pairs. We speculate that two high-frequency modes (1588 and 1596 cm^-1) are involved in the vibronic coupling leading to fast (<ps) and direct energy transfer from the highest to lowest exciton, bypassing the intermediate excitons.

Figure 1

(a) Crystal structure of PC645 (4lms) shows the spatial arrangement of the biliverdin pigments. (b) Chemical structures of the three main biliverdin pigments: dihydrobiliverdin (DBV), mesobiliverdin (MBV), and phycocyanobilins (PCB). (c) Linear absorption spectrum of PC645 at 120 K is represented in a black solid line. Three different excitation spectra (centered at 16 250, 15 625, and 14 925 cm^–1, respectively) are used for excitation-frequency-dependent pump-probe experiments, shown with different filled curves. The excitonic peak maxima are indicated by colored vertical lines according to literature values: PCB82 (orange), PCB158 (yellow), MBV (green), DBV–, and DBV+ (blue).

Figure 2

(a–c) Transient IR spectra with increase (blue to orange) in pump-probe delays (0.2, 0.5, 1, 2, 5, 10, and 50 ps) observed with the three different excitation spectra centered at 16 250, 15 625, and 14 925 cm^–1 shown in Figure 1c. (d–f) Time evolutions of the IR bands at 1588, 1596, 1619, 1653, 1681, and 1732 cm^–1 are shown in black, blue, green, yellow, red, and magenta, respectively.

Figure 3

Evolution-associated difference spectra obtained from the global analysis of the transient IR spectra obtained with three different excitation spectra shown in Figure 1c. The GSB peaks at 1588 and 1596 cm^–1 peaks are marked with green and yellow arrows, respectively, to highlight the change in their relative intensities.

Figure 4

2DEV spectra of PC645 at 120 K at four selected waiting times T = 100 fs, 200 fs, 2 ps, and 10 ps. The dotted vertical lines represent the excitonic peak maxima. The top panel of each 2D graph shows the normalized linear absorption spectrum (black) of PC645 at 120 K and the broadband excitation spectrum (brown) covering all of the excitons shown with colored vertical lines. The excitonic peak maxima are indicated by the colored solid vertical lines: PCB82 (orange), PCB158 (yellow), MBV (green), DBV–, and DBV+ (blue).

Figure 5

2DEV spectra at the excitation frequencies corresponding to the exciton peak maxima (a) DBV (16 750 cm^–1), (b) MBV (16 130 cm^–1), (c) PCB158 (15 800 cm^–1), and (d) PCB82 (15 270 cm^–1) at four waiting times (top to bottom: 100 fs, 200 fs, 2 ps, 10 ps). The vertical gray dotted lines represent the IR bands that commonly appear in all excitons. The orange dotted lines in (a) represents the marker band for PCB82. The IR bands that show major change in spectral features with an increase in waiting time are highlighted with colored arrows. The blue, green, yellow, orange, and red colors indicate the characteristic IR bands for DBV, MBV, PCB158, PCB82-D, and PCB82-C, respectively.

Figure 6

Schematic representation of the interpigment energy flow in PC645 (time constants within arrows, in picoseconds), as reported by Marin et al. with the marker infrared signatures specific to each pigment we reported in this study (bold italic number within boxes, in wavenumbers). Only hydrogen bonds with side chain amino acids are shown (PDB: 4lms).