Effects of dehydration on reaction centers from Rhodopseudomonas sphaeroides

Air-dried films of reaction centers from Rhodopseudomonas sphaeroides were found to resemble aqueous suspensions of these reaction centers in their optical and photochemical properties, except that the long wave absorption band of bacteriochlorophyll was shifted from about 860 to 845 nm. The quantum efficiency of the photochemical reaction that produces oxidized bacteriochlorophyll and reduced ubiquinone was greater than 0.75 in the films, using 800-nm actinic light. Dehydration of the films caused further blue shifts of all the absorption bands between 500 and 900 nm, and some loss (about 20%) of intensity of the long wave band, coupled to a gain of absorbance centered at 795 nm. The latter changes were not accompanied by an increase at 1250 nm, ruling out the oxidation of bacteriochlorophyll as a cause of these changes. Dehydration caused about 50% of the reaction centers to become photochemically inactive. For the component that remained active, the photochemical quantum efficiency was about 0.5 at room temperature, rising to 0.7 or more as the temperature was brought below 250 K. The yield of 900 nm fluorescence was approximately the same in air-dried films as in aqueous suspensions; dehydration of a film raised the fluorescence yield about 3-fold. The yield of this fluorescence in a dehydrated film was independent of temperature (+/- 10%) between 300 and 70 K. The back reaction after illumination, return of electrons from reduced ubiquinone to oxidized bacteriochlorophyll, has kinetics in which a most rapid first-order component is mixed with slower components. The fastest component, identified with the return of electrons from 'primary' ubiquinone to oxidized bacteriochlorophyll, was predominant in the materials tested here. Its half time in an aqueous suspension of reaction centers fell from 86 ms at 300 K to 33 MS AT 200 K and declined more gradually to 15 ms at 50 K. An air-dried film showed similar behavior. The dramatic change of half-time between 300 and 200 K can be ascribed to one or more phase transitions involving water; in a dehydrated film the half-time of the fastest decay component was about 22 +/- 5 ms, independent of the temperature between 300 and 70 K. The original properties of an air-dried film were restored fully, after dehydration, by exposure to either H2O or 2H2O vapor. There was no significant difference, in these measurements, between H2O-restored and 2H2O-restored films.