Femtosecond Stimulated Raman Study of Energy Transfer in Allophycocyanin

Abstract

Allophycocyanin (APC) is a light-harvesting protein found in cyanobacteria and red algae. APC along with other phycobiliproteins such as phycocyanin (PC) and phycoerythrin (PE) constitutes phycobilisome and plays an important role in collecting light and cascading the excitation energy into a reaction center where the light energy is converted into chemical potential. Thus it is important to understand how excitation energy is transferred between the phycobiliproteins and inside them. APC is a trimer of αβ subunit, each of which contains a linear tetrapyrrole chromophore, phycocyanobilin (PCB), covalently bound to a cystein residue (Fig. 1). Close proximity (~20 A) between α-84 and nearby β-84 in APC can cause the electronic states of the chromophores to interact to form split exciton states. In this case, interexciton dynamics mediates the energy transfer. On the other hand, it is also possible that energy transfer from α-84 to β-84 occurs via dipole-dipole coupling between the two chromophores (Forster model). The intermediate range of distance between the PCBs in APC makes both models quite feasible for an energy transfer mechanism and has generated much controversy. Here we report on the excited state dynamics of APC measured by femtosecond stimulated Raman spectroscopy. Femtosecond stimulated Raman spectroscopy (FSRS) reveals the structure of fast-evolving molecules by providing vibrational spectra with excellent spectral resolution on a femtosecond time scale. We prepare the electronic excited state of APC by 40 nJ, 30 fs actinic pump pulse at 1 kHz, centered at 610 nm. The femtosecond stimulated Raman spectra of the excited state APC are obtained at a series of time delays by coupling 0.5 μJ/pulse, 2 ps, 800 nm Raman pump pulse and 4 nJ/pulse, 20 fs, broadband continuum Raman probe pulse. The detailed description of the detection system and how to generate the laser pulses has been published elsewhere. APC was isolated from the filamentous cyanobacterium Anabaena variabilis and used in phosphate buffer solution. Figure 2 presents the FSRS spectra of the ground state and the excited state APC at selected time delays. The excited state spectra were obtained by subtracting the spectra of buffer solution and the ground state APC which accounts for the unexcited 85% of APC. The ground state FSRS spectrum is in excellent agreement with the resonance Raman spectra previously reported at UV wavelengths. The excited state spectra show broad and dispersive features without any well-defined peaks. The amplitude of the dispersive features increases up to ~80 fs and decreases gradually, completely gone by 5 ps (not shown). McCamant et al. found that dispersive lineshapes appear when the Raman pump and probe pulses drive resonant stimulated emission from the excited state and create vibrational coherence on the electronic ground state surface, which they termed the Raman initiated by nonlinear emission (RINE). The RINE spectra contain the information on the vibrational frequencies of the electronic ground state whose dynamics is determined by the evolution of the excited state population. From this model, McCamant et al. successfully extracted the vibrational spectra of the ground state bacteriorhodopsin, which evolve with the population changes in the excited state. Adopting a similar method described in ref. 14, we fit the excited state FSRS spectra of APC to the RINE model. From