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

Sodium-Dependent Movement of Covalently Bound FMN Residue(s) in Na⁺-Translocating NADH:Quinone Oxidoreductase

Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na⁺ potential. We found that the change in Na⁺ concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na⁺-NQR from <i>Vibrio harveyi</i>, as was revealed by the anaerobic equilibrium redox titration of the enzyme’s EPR spectra. On the other hand, the change in Na⁺ concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN_NqrB and FMN_NqrC). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN_NqrB and FMN_NqrC was found to be 15.3 Å in the absence and 20.4 Å in the presence of Na⁺, respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 Å upon changes in Na⁺ concentration. Using these results, we propose a scheme of the sodium potential generation by Na⁺-NQR based on the redox- and sodium-dependent conformational changes in the enzyme