Phototraping of doubly reduced monomeric bacterichlorophyll in the photoreaction center of Ectothiorhodospira sp.

Abstract

The photoreaction center from the purple sulfur bacterium Ectothiorhodospira sp. was illuminated in the presence of reduced cytochrome c or dithionite under anaerobic conditions. This treatment first caused the monoelectronic reduction of both molecules of bacteriopheophytin (Bph), phi A and phi B, as witnessed by the appearance of EPR and optical signals typical of singly-reduced bacteriochlorins. Continued illumination under the same reducing conditions caused both of these signals to disappear. Such disappearance was accompanied by a complete bleaching of the Qx and Qy absorption bands of Bph but not of the corresponding transitions of bacteriochlorophyll (Bchl). These phenomena are interpreted by a double reduction of phi A and phi B. As long as the medium remained reducing and anaerobic, these changes were stable. Prolonged illumination under the same reducing conditions finally led to the bleaching of the Qx (600 nm) and Qy (800 nm) bands of Bchl but not of the 880-nm band. This generated no EPR or 645-nm absorption signals due to singly-reduced Bchl. The bleaching kinetics of the 800-nm band was biphasic and paralleled a shift of the peak wavelength. This is interpreted by a double reduction of both molecules of monomeric Bchl BA and BB in an undetermined order. After bleaching of the 800-nm band has reached saturation, the absorbance ratio of the 800/880-nm absorption bands remains constant, as would be expected if the ultimate spectrum was that of the primary electron donor. These experiments demonstrate the photoreduction of Bchl and allow the absorption spectrum of the primary donor to be measured for the first time.