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

Alexander V. Bogachev,Marina L. Verkhovskaya,Mahir D. Mamedov,Yulia V. Bertsova,Vladimir P. Skulachev

doi:10.1038/srep21397

Alexander V. Bogachev, Marina L. Verkhovskaya + Show 3 more

Open Access

https://doi.org/10.1038/srep21397

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Abstract

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 → K585 → (L450↔M495) → O585 → 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 4×104 s–1, 4.7 × 103 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.

Highlights

We studied kinetics of the electrogenic response of the Na+-pumping rhodopsin from Dokdonia sp
Due to the good match between the kinetics of intermediates formation in the NaR photocycle and electric potential generation (Fig. 7A), it can be concluded that the phases I, II, and III of Δ ψ formation correspond to the K → (L↔ M), (L↔ M) → O, and O → NaR transitions, respectively
PRO95 is highly similar to the well-studied NaR of K. eikastus NBRC 100814T (98% identity)

Summary

Introduction

The three-dimensional structure of NaR from K. eikastus NBRC 100814T was determined by X-ray structural analysis[11,12] This approach revealed a number of structural properties specific for NaR that are absent from other retinal-dependent ion pumps[11,12]. Direct electrometry is a convenient method for detecting transmembrane transfer of charged species by proteins operating as generators of transmembrane electric potential (Δ ψ ) This approach has been successfully used in studies of bacterial photosynthetic reaction centers and cytochrome bc1-complex[13,14,15], pigment–protein complexes of photosystems II and I16,17, terminal oxidases[18,19,20,21], bacteriorhodopsin[22,23], and halorhodopsin[24]. We used this method to study the catalytic cycle of the recently described NaR

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