Spectral and temporal differentiation between integral and contaminant chlorophyll a in the cytochrome b₆f complex

Purification of photosynthetic protein complexes in detergent often results in residual contaminant chlorophyll (Chl) that is dependent on the preparation process. In the case of the cytochrome b₆f complex, which contains one molecule of bound Chl a per 130 kDa monomer in the dimeric hetero-oligomeric complex, both complex-bound and contaminant Chl a are present and spectrally indistinguishable in the steady-state absorbance spectra commonly employed to assay photosynthetic protein complexes. We, however, demonstrate that the signals from photo-excited cyt b₆f-bound and contaminant Chl a have distinct temporal and spectral signatures, as revealed by ultrafast optical transient spectroscopy. The difference in signals is further amplified by using a non-anisotropic pump-probe scheme to enhance the detection of the contaminant Chl a stimulated emission. Such sharp differences further exemplify the impact of the environment on the photodynamic properties of molecules.