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.