Role of the PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 2. PufX is required for efficient ubiquinone/ubiquinol exchange between the reaction center QB site and the cytochrome bc1 complex.

Abstract

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., Verméglio, 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 bc1 complex: When the kinetics of flash-induced reduction of cytochrome b561 were monitored in chromatophores, we observed a requirement of PufX for turnover of the cytochrome bc1 complex only at high redox potential (Eh > 140 mV), suggesting a function of PufX in lateral ubiquinol transfer from the RC. In 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 acceptors 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/QH2 exchange at the QB site of the RC. We show that the Q/QH2 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 QB 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.

Functional Consequences of the Organization of the Photosynthetic Apparatus in Rhodobacter sphaeroides

“…We thus again observed a "significant but moderate" effect of the PufX deletion. Similar experiments were reported by Barz et al (18) and Francia et al (15), who found a larger slowing (Ͼ10-fold) and a pronounced lag of ϳ9 ms before the onset of the reduction kinetics. In our experiments, the time course of heme b reduction is sigmoidal, with a lag of only ϳ2 ms. We do not know the origin of this discrepancy.…”

“…Moreover, this barrier exerts a significant but moderate effect, slowing the turnover rate by slightly Ͼ2-fold. This is far from the dramatic blocking suggested by previous reports (17,18), but it makes the photosynthetic competence of other bacteria with a native closed LH1 ring easier to understand.…”

“…Such mutants have lost the ability to grow photosynthetically under anaerobic reducing conditions. Photosynthetic growth is recovered, however, when oxidants such as trimethylamine N-oxide and Me 2 SO are added to the medium (17,18). When examined on a single turnover basis, the electron transfer kinetics appear normal.…”

“…When examined on a single turnover basis, the electron transfer kinetics appear normal. However, a dramatic slowing down of the cyclic flow is observed under steadystate illumination under anaerobic conditions (17,18). It was suggested that the closed LH1 ring around the RC obstructs the shuttling of quinol/quinone between the RC and bc 1 complexes.…”

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX −

“…It has been suggested that a protein found in both Rhodobacter sphaeroides and Rhodobacter capsulatus called PufX may provide an answer to this question (10). PufX appears to be a member of the LH1 ring, replacing one of the ␣␤ dimers.…”