Real-time kinetics of electrogenic Na(+) transport by rhodopsin from the marine flavobacterium Dokdonia sp. PRO95

Discovery of the light-driven sodium-motive pump Na(+)-rhodopsin (NaR) has initiated studies of the molecular mechanism of this novel membrane-linked energy transducer. In this paper, we investigated the photocycle of NaR from the marine flavobacterium Dokdonia sp. PRO95 and identified electrogenic and Na(+)-dependent steps of this cycle. We found that the NaR photocycle is composed of at least four steps: NaR(519) + hv → K(585) → (L(450)↔M(495)) → O(585) → NaR(519). The third step is the only step that depends on the Na(+) concentration inside right-side-out NaR-containing proteoliposomes, indicating that this step is coupled with Na(+) binding to NaR. For steps 2, 3, and 4, the values of the rate constants are 4×10(4) s(–1), 4.7 × 10(3) M(–1) s(–1), and 150 s(–1), respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Δψ ~ 200 mV) generated by a single turnover of NaR incorporated into liposomes and attached to phospholipid-impregnated collodion film. On the basis of these observations, a mechanism of light-driven Na(+) pumping by NaR is suggested.