The Quenching of Long-Wavelength Fluorescence by the Closed Reaction Center in Photosystem I in Thermostichus vulcanus at 77 K

Abstract

Photosystem I in most organisms contains long-wavelength or "Red" chlorophylls (Chls) absorbing light beyond 700 nm. At cryogenic temperatures, the Red Chls become quasi-traps for excitations as uphill energy transfer is blocked. One pathway for de-excitation of the Red Chls is via transfer to the oxidized RC (P700⁺), which has broad absorption in the near-infrared region. This study investigates the excitation dynamics of Red Chls in Photosystem I from the cyanobacterium Thermostichus vulcanus at cryogenic temperatures (77 K) and examines the role of the oxidized RC in modulating their fluorescence kinetics. Using time-resolved fluorescence spectroscopy, the kinetics of Red Chls were recorded for samples with open (neutral P700) and closed (P700⁺) RCs. We found that emission lifetimes in the range of 710-720 nm remained unaffected by the RC state, while more red-shifted emissions (>730 nm) decayed significantly faster when the RC was closed. A kinetic model describing the quenching by the oxidized RC was constructed based on simultaneous fitting to the recorded fluorescence emission in Photosystem I with open and closed RCs. The analysis resolved multiple Red Chl forms and variable quenching efficiencies correlated with their spectral properties. Only the most red-shifted Chls, with emission beyond 730 nm, are efficiently quenched by P700⁺, with rate constants of up to 6 ns^-1. The modeling results support the notion that structural and energetic disorder in Photosystem I can have a comparable or larger effect on the excitation dynamics than the geometric arrangement of Chls.

Keywords: Thermosynechococcus vulcanus; chlorophyll; cyanobacteria; energy transfer; light harvesting; time-resolved fluorescence.

Figure 1

The time-resolved fluorescence of PSI in T. vulcanus at 77 K. (A) The fluorescence decay kinetics at four emission wavelengths from 690 nm to 750 nm. The symbols indicate the measured data points, and the lines are obtained by global multiexponential fitting. RC_red represents a sample containing ≈50% open (reduced) RCs, and RC_ox represents a sample with closed (oxidized) RCs. The decays are normalized at the respective global maximum and plotted on a semi-logarithmic scale. (B) The time-gated fluorescence spectra at four decay times from 0.1 to 2 ns. The symbols and lines represent data and fit, respectively.

Figure 1

The time-resolved fluorescence of PSI in T. vulcanus at 77 K. (A) The fluorescence decay kinetics at four emission wavelengths from 690 nm to 750 nm. The symbols indicate the measured data points, and the lines are obtained by global multiexponential fitting. RC_red represents a sample containing ≈50% open (reduced) RCs, and RC_ox represents a sample with closed (oxidized) RCs. The decays are normalized at the respective global maximum and plotted on a semi-logarithmic scale. (B) The time-gated fluorescence spectra at four decay times from 0.1 to 2 ns. The symbols and lines represent data and fit, respectively.

Figure 2

The decay-associated fluorescence emission spectra of isolated PSI complexes obtained from a global analysis of fluorescence decays recorded at 77 K with 440 nm excitation. (A) PSI with open and closed RCs (≈50% P₇₀₀⁺) and (B) PSI with closed RCs (P₇₀₀⁺). The spectra are normalized to their maxima with the relative normalization factors shown in parentheses.

Figure 3

The wavelength dependence of the amplitude-weighted average fluorescence lifetime ${\tau }_{av}={\sum }_i{a}_i{\tau }_i/{\sum }_i{a}_i$ at 77 K, compared for the open and closed RCs. Note that decay components with an ≈5-ns lifetime attributed to free Chls are excluded from the calculation.

Figure 4

The heterogeneous target analysis of PSI kinetics at 77 K. (A) The kinetic model schemes of PSI with a closed RC (P₇₀₀⁺) with energy transfer and trapping rate constants in ns⁻¹. With an open RC, the decay rates of Red 2b and Red 2c are both 0.2 ns⁻¹. (B) The species-associated emission spectra (SAES), including an unconnected compartment “Add”. The amplitude of the “Bulk” SAES is multiplied by 5 for readability. (C) The species transient populations (time-dependent concentrations). The solid/dashed lines represent open/closed RCs. Note that the horizontal scale is linear until 100 ps and logarithmic thereafter.

Figure 5

The time traces of the PSI emission at two wavelengths (indicated in the title of the panels) after 440 nm excitation at 77 K. The grey (orange) and black (red) lines indicate the data and the target analysis fit of the PSI in the closed and “open” RC experiments, respectively. Note that the time axis is linear until 100 ps and logarithmic thereafter. Note also that each panel is scaled to its maximum. The overall rms error of the fit was 18.3.

Figure 6

An illustration of the quenching of Red Chl emission in T. vulcanus’s PSI based on the structure and tentative assignment of Chls contributing to the low-energy states. The Chl positions are drawn using PDB 1JB0 [2]. Red Chls are denoted with yellow and red colors. The curved dashed arrows represent multistep energy transfer to the RC via intermediate-energy bulk Chl states. The straight solid arrows represent direct Förster resonance energy transfer to the oxidized RC (P₇₀₀⁺). The transfer lifetimes can vary depending on the (inhomogeneously distributed) Red Chl energies. The intermediate-energy state contributed by Chls A₃₂/B₇ is de-trapped mainly by uphill energy transfer through the bulk Chls. The low-energy state contributed by Chls B₃₁/B₃₂/B₃₃ is quenched by direct transfer to P₇₀₀⁺.