Rhodopsin from Haloquadratum walsbyi is a light-driven magnesium transporter.

The functionally unknown Middle rhodopsin (HwMR) is a microbial rhodopsin (mRho) identified in Haloquadratum walsbyi, an archaeon that thrives in a 2 M MgCl environment harmful to most other microorganisms. HwMR shares conserved and functionally critical residues with both bacteriorhodopsin (BR), a proton pump, and sensory rhodopsin II (SRII), which mediates phototaxis, even though HwMR exerts neither function. We previously reported HwMR as a unique mRho found to associate with Mg. Here, we show that HwMR can sense environmental Mg concentration via the D84 residue according to characteristic maximum absorption wavelength shift, photocycle kinetics, and Mg titration assay. X-ray crystallography of the wild-type HwMR and its D84N mutant produced two HwMR atomic structure models. Omit maps analysis of the wild-type HwMR model revealed D84 as a Mg binding site. On the cytoplasmic side, omit maps also revealed Mg association with T216. Both Mg sites were absent in the D84N mutant. A cell-based light-driven conductivity assay provided evidence to propose that HwMR is an inward magnesium transporter, with D84 as the primary binding site and T216 as the transportation stabilizing site. A sequential model was proposed to illustrate Mg transportation in HwMR.