Continuum probe two-dimensional electronic spectroscopy of the photosystem II reaction center

We report two-dimensional electronic spectroscopy of the photosystem II reaction center, collected in the pump-probe geometry employing a continuum probe. This enables observation of ion bands that report on intermediates in the charge

selectively exciting across the Q y band (660-690 nm) with a narrowband pump [9].In these studies a broadband probe enabled the indentification of charge separation intermediates, leading to the proposal that two distinct charge separation pathways are active at 77 K.We have previously performed 2DES on the Q y band of the PSII RC [10], directly observing excitonic coupling in the system, and mapping the exchange of energy among excitonic states with high time resolution.In this study we extend the spectral window of our previous measurements, collecting 2DES of the PSII RC with a continuum probe spanning 450-700 nm.Of critical interest is a relatively weak 455 nm feature associated with a pheophytin anion.As a direct signature of intermediate charge separation, this feature can be used to correlate transient features in the Q y band with the charge separation process.
A home-built non-colinear optical parametric amplifier (NOPA) powered by a 1kHz commercial chirped pulse amplifier (Spectra Physics Spitfire Pro) produces our tunable pump source.The pair of collinear pump pulses with variable relative time delay required for 2D spectroscopy in the pump probe geometry are created from the NOPA beam using an acousto-optic pulse shaper (Fastlite Dazzler).The pump pulses are also temporally compressed to 30 fs by the pulse shaper.A broadband probe is generated by launching a 800 nm 35fs pulse into a CaF 2 crystal, which is kept in constant planar motion to prevent damage to the crystal.To avoid lineshape and kinetic distortions resulting from chirp in the probe we compress the continuum to 30 fs using a pair of extremely broadband chirped mirrors (Layertec) with approximately -40 fs 2 GVD per bounce.Population kinetics are observed by altering the relative delay between pump and probe pulses.The entire detection axis is dispersed onto a cooled CCD (Princeton Pixis) using a 300 groove/mm grating (Horiba iHR320) and read out at the laser repetition rate.Noise resulting from probe fluctuations is removed by simultaneously collecting an unpumped reference pulse in the same spectrometer.We employ a four phase cycle detection scheme with probe chopping to remove pump scatter and transient absorption artifacts.
PSII reaction center complexes were prepared from spinach using a slightly modified version of van Leeuwen's protocol [11], where the ratio of peak amplitudes in the Soret band was used as a measure of purity.After elution from the column, samples were centrifuged with membrane filters to remove excess salt and increase concentration.Glycerol was added to the samples in Bis-Tris buffer pH 6.5 (67%/33% v/v) to act as a cryoprotectant.Samples were cooled to 77K in a cryostat (Oxford Instruments Microstat N). and bottom rows).Contours here are on a linear scale (10% per contour for the top row of figures, and 2.5% per contour for the bottom row).The signal levels for the top row are as much as 100 times weaker, but features from pheophytin, carotenoids, and chlorophyll are present.
Data was collected over waiting times ranging from 0-180 ps to monitor the appearance and decay of the weak high energy features.Consistent with pump probe studies [9], we find that the 455 nm (660 THz) pheophytin anion signature is appreciable by ~3 picoseconds.We also observe other transitions of interest, including the chlorophyll a Q x and Q y (1,0), pheophytin Q x and carotenoid bands.In our previous Q y studies, we identified the spectral signatures of different kinetic processes using "two dimensional decay associated spectra" (2D DAS).The 2D DAS revealed distinct spectral features on the 1-3 ps timescale that has been associated with the primary charge separation process.The additional information available from the continuum probe studies presented here will enable direct correlations to be made between the spectral changes in the Q y band and charge separation events.Also ongoing are efforts to simulate the 2DES data based on current excitonic models [8].The more extensive data set provided by continuum probe 2DES will allow more rigorous testing and refinement of these models.

Fig 1 :
Fig 1: Two dimensional absorptive spectrum of the d1d2 cyt-b559 PSII RC at 77 K for waiting times of 200 fs (left) and 100 ps (right) in two different frequency regions (top