Bacterial Na+-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na+, but some can additionally transport Hi To determine the structural basis of cation promiscuity in Na+-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na+-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na+-rhodopsin (Kr2). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na+-rhodopsin (NaR) was indeed found to transport both Na+ and H+ in a light dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation capture step by comparison with the wild-type NaR in a Na+-deficient medium. The structural basis for the dependence of the Na+/H+ specificity in Na+-rhodopsin on residue 253 remains to be determined. (C) 2018 Elsevier Inc. All rights reserved.