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: NaR519 + hv -> K-585 -> (L-450 M-495) -> O-585 -> NaR519. 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 4x10(4) s(-1), 4.7 x 10(3) M-1 s(-1), and 150 s(-1), respectively. These steps contributed 15, 15, and 70% of the total membrane electric potential (Delta psi similar to 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.Peer reviewe

Translocation of electrical charge during a single turnover of cytochrome-c oxidase

AbstractIn cell respiration, cytochrome-c oxidase utilizes electrons from catabolism to reduce O2 to water. Energy is conserved as an electrochemical proton gradient across the mitochondrial membrane, which drives the synthesis of ATP. Electrical charge translocation during the reaction of the reduced enzyme with O2 takes place in two phases of identical amplitude. The first phase (τ1=0.2 ms) occurs after an initial lag, and appears to correspond to the transition from a peroxy to a ferryl intermediate in the oxygen chemistry. The second phase (τ2=2.6 ms) matches the transition from the ferryl intermediate to the oxidised enzyme. These findings define the kinetic linkage between the chemistry and the major events of proton pumping by the enzyme