Characterization of an LM unit purified by affinity chromatography from Rhodobacter sphaeroides reaction centers and interactions with the H subunit

Abstract

Reaction centers from wild-type Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides) were separated into two components: the LM complex and H subunit. LM was isolated after brief treatment of reaction centers with SDS by affinity chromatography with cytochrome c as ligand. A stable H preparation was obtained after dissociation of reaction centers with lithium perchlorate. LM was depleted of the transition metal, Mn, which interacts with Q A and Q B in native reaction centers. It retained only 30% of primary photochemistry which could be restored to 50–80% by addition of Q 6, Q 10 or other quinones. A stable semiquinone radical Q − A could be flash-induced in LM. Its absorption properties are similar to those of Q − A in native reaction centers. The quantum yield of photochemistry in an LM unit reconstituted with Q 6 is the same as in intact reaction center and in LM in the presence of H. This result was confirmed by the rapid electron-transfer rate between I − and Q A in LM + H ( τ ≈ 0.45 ns). Ubiquinone in LM incubated with H becomes tightly bound at the Q A site. Flash production of a Q 2− species was not detected in LM and LM + H. We conclude that the depletion of the reaction center both of the H subunit and of the metal does not necessarily lower the quantum yield of the primary reaction or greatly modify the rate of electron transfer from I − to Q A. These results contrast with observations of others that seemed to demonstrate that the metal is essential for high-rate electron transfer between I − and Q A (Debus, R.J., Feher, G. and Okamura, M.Y. (1986) Biochemistry 25, 2276–2287). In our experiments, secondary electron transfer to Q B was not restored in LM + H, unlike in reconstitution experiments reported with R26 Rb. sphaeroides reaction centers (Debus, R.J., Feher, G. and Okamura, M.Y. (1985) Biochemistry 24, 2488–2500). Apparently, interactions between H and LM were too weak for restoring Q B activity.