LOCALIZATION OF THE PRIMARY QUINONE BINDING-SITE IN REACTION CENTERS FROM RHODOPSEUDOMONAS-SPHAEROIDES R-26 BY PHOTOAFFINITY-LABELING

We have prepared a radioactive photoaffinity label, 2-azido[3H]anthraquinone, to substitute for ubiquinone as the primary electron acceptor in reaction centers from the pho-tosynthetic bacterium Rhodopseudomonas sphaeroides R-26. When the label was illuminated with ultraviolet light, it photolyzed to yield an intermediate, most likely a triplet nitrene, which was observed at 80 K by optical and EPR spectroscopy. Reacton centers that had the ubiquinone replaced by the label showed photochemical activity (bleaching at 865 nm and light-induced EPR signals) at room temperature, 80 K, and 2.1 K. When reaction centers reconstituted with the label were illuminated with ultraviolet light at 80 K and subsequently warmed, some of the label became covalently attached to the protein. Similar results were obtained with infrared illumination, showing that nitrene formation can be mediated by the tetrapyrrole pigments. Analysis of photolyzed protein samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the M subunit of the reaction center was selectively labeled, as compared to control preparations in which the primary quinone binding site was filled by ubiquinone before labeling. These results show that the primary quinone binding site is located on or very close (within ~5 Å) to the M subunit of the reaction center. © 1979, American Chemical Society. All rights reserved.