TRYPSIN TREATMENT OF REACTION CENTERS FROM RHODOBACTER-SPHAEROIDES INTHE DARK AND UNDER ILLUMINATION - PROTEIN STRUCTURAL-CHANGES FOLLOW CHARGE SEPARATION

Reaction centers from Rhodobacter sphaeroides R-26 were treated with t rypsin in the dark and during illumination (in the charge-separated st ate). Trypsination resulted in a time-dependent modification of the re action centers, reflected in changes in the charge recombination rate, in the inhibition of Q(A)(-) to Q(B) electron transfer, and eventuall y to inhibition of charge separation. Comparisons of centers with ubiq uinone or anthraquinone in the Q(A) site, in which the charge recombin ation pathways are different, indicate that trypsination affects charg es close to the Q(A)(-)-binding site. Studies of light-induced voltage changes from moving charges in reaction centers incorporated in lipid layers on a Teflon film, a technique which allows the discrimination of effects on donor and acceptor sides, indicate that the acceptor sid e is preferentially degraded by trypsin in the dark. Tryptic digestion during illumination generally resulted in a marked strengthening and acceleration of the effects seen already during dark treatment, but ne w effects were also detected in gel electrophoretic peptide patterns, in optical spectra, and in the kinetic measurements. Optical kinetic m easurements revealed that the donor side of the reaction centers becam e susceptible to modification by trypsin during illumination as seen i n the value of the binding constant for soluble cytochrome c(2) which increased by a factor of 2, whereas it was much less affected after tr ypsination of reaction centers in the dark. The influence of illuminat ion on the rate and mode by which trypsin acts on reaction centers ind icates that changes in the protein conformation follow charge separati on. The function of these light-induced structural changes may be to s tabilize charge separation and facilitate forward electron transfer.