ROLE OF THE PUFX PROTEIN IN PHOTOSYNTHETIC GROWTH OF RHODOBACTER-SPHAEROIDES .2. PUFX IS REQUIRED FOR EFFICIENT UBIQUINONE UBIQUINOL EXCHANGE BETWEEN THE REACTION-CENTER Q(B) SITE AND THE CYTOCHROME BC(1) COMPLEX

The PufX membrane protein is essential for photosynthetic growth of Rhodobacter sphaeroides because it is required for multiple-turnover electron transfer under anaerobic conditions [see accompanying article; Barz, W. P., Francia, F., Venturoli, G., Melandri, B. A., Vermeglio, A., & Oesterhelt, D. (1995) Biochemistry 34, 15235-15247]. In order to understand the molecular role of PufX, light-induced absorption spectroscopy was performed using a pufX(-) mutant, a pufX(+) strain, and two suppressor mutants. We show that the reaction center (RC) requires PufX for its functionality under different redox conditions than the cytochrome bc(1) complex: When the kinetics of flash-induced reduction of cytochrome b(561) were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome b(c1) complex only at high redox potential (E(h) > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. Tn contrast, PufX is required for multiple turnover of the RC only under reducing conditions: When the Q pool was partially oxidized in vivo using oxygen or electron accepters like dimethyl sulfoxide or trimethylamine N-oxide, the deletion of PufX had no effect on light-driven electron flow through the RC. Flash train experiments under anaerobic in vivo conditions revealed that RC photochemistry does not depend on PufX for the first two flash excitations. Following the third and subsequent flashes, however, efficient charge separation requires PufX, indicating an important role of PufX for fast Q/QH(2) exchange at the Q(B) Site of the RC. We show that the Q/QH(2) exchange rate is reduced approximately 500-fold by the deletion of PufX when the Q pool is nearly completely reduced, demonstrating an essential role of PufX for the access of-ubiquinone to the Q(B) site. The fast ubiquinone/ubiquinol exchange is partially restored by suppressor mutations altering the macromolecular antenna structure. These results suggest an indirect role of PufX in structurally organizing a functional photosynthetic apparatus.