Excitation energy transfer in Rhodobacter sphaeroides analyzed by the time-resolved fluorescence spectroscopy

Abstract

Excitation energy transfer in the photosynthetic bacterium, Rhodobacter sphaeroides, was analyzed using time-resolved fluorescence spectroscopy measured with a new type of microchannel-plate photomultiplier (S-l type photocathode). Difference spectra and Gaussian deconvolution of fluorescence spectra of the bacterium at −196°C clearly revealed three fluorescence components (F875, F911, F925). These were attributed to B850, B875 and an additional longer-wavelength antenna component (B890), respectively. Emission from B800 was not detected, indicating a very fast energy transfer from B800 to B850 with a transfer time shorter than 6 ps. Similar measurements also suggested the same upper limit for the time of energy transfer from B850 to B875. The rise and decay kinetics of the fluorescence components indicate a sequential energy flow in the order of B800, B850, B875, B890 and finally the reaction center. The three fluorescence components (F859, F889, F909) were also detected in spectra taken at 22°C and were attributed to B850, B875 and B890, respectively. Changes in the fluorescence spectra were observed only in the initial 100 ps afterthe excitation pulse. The time needed for apparent energy transfer between components was estimated from the rise term of kinetics; 20 ps for B850 to B875 and 35 ps for B875 to B890. A rise in the relative intensity of F909 in the time-resolved spectra suggested a 50 ps excitation equilibration time between B890 and the reaction center. In a later time range, no spectral changes were observed, indicating equilibration of excitation transfer among the antenna components. After equilibration, the main fluorescence component decayed with the lifetime of 255 ± 10 ps. Based on the deconvoluted spectra, the absorption maximum of the longer-wavelength antenna was estimated to be 890 nm at 22°C and 898 nm at −196°C. The relative intensities of the deconvoluted fluorescence bands were used to estimate the content of B890 bacteriochlorophyll to be 7 ± 1 per reaction center.