Microbial rhodopsins (MRho) are vital proteins in Haloarchaea for solar light sensing in extreme living environments. Among them, Haloquadratum walsbyi (Hw) is a species known to survive high MgCl₂ concentrations, with a total of three MRhos identified, including a high-acid-tolerance light-driven proton outward pump, HwBR, a chloride-insensitive chloride pump, HwHR, and a functionally unknown HwMR. Here, we showed that HwMR is the sole magnesium-sensitive MRho among all tested MRho proteins from Haloarchaea. We identified at least D84 as one of the key residues mediating such magnesium ion association in HwMR. Sequence analysis and molecular modeling suggested HwMR to have an extra H8 helix in the cytosolic region like those in signal-transduction-type MRho of deltarhodopsin-3 (dR-3) and Anabaena sensory rhodopsin (ASR). Further, HwMR showed a distinctly prolonged M-state formation under a high concentration of Mg²⁺. On the other hand, an H8 helix truncated mutant preserved photocycle kinetics like the wild type, but it led to missing M-state structure. Our findings clearly suggested not only that HwMR is a novel Mg²⁺-associated protein but that the association with both Mg²⁺ and the H8 domain stabilizes M-state formation in HwMR. We conclude that Mg²⁺ association and H8 are crucial in stabilizing HwMR M state, which is a well-known photoreceptor signaling state.

微生物视紫红质 (MRho) 是盐古菌中重要的蛋白质，用于极端生活环境中的太阳光传感。其中，Haloquadratum walsbyi (Hw) 是已知能够在高 MgCl ₂浓度下生存的物种，总共鉴定了 3 个 MRho，包括高耐酸光驱动质子外向泵、HwBR、对氯不敏感的氯离子泵、 HwHR 和功能未知的 HwMR。在这里，我们证明 HwMR 是所有测试的盐古菌 MRho 蛋白中唯一的镁敏感 MRho。我们至少鉴定出 D84 是 HwMR 中介导此类镁离子缔合的关键残基之一。序列分析和分子模型表明，HwMR 在胞质区域有一个额外的 H8 螺旋，就像 deltahodopsin-3 (dR-3) 和鱼腥藻感觉视紫红质 (ASR) 的信号转导型 MRho 中的螺旋一样。此外，HwMR在高浓度Mg ²⁺下显示出明显延长的M态形成。另一方面，H8螺旋截短的突变体保留了与野生型一样的光循环动力学，但它导致了M态结构的缺失。我们的研究结果清楚地表明，HwMR 不仅是一种新型 Mg ^{2+相关蛋白，而且与 Mg 2+}和 H8 结构域的关联稳定了 HwMR 中 M 态的形成。我们得出结论，Mg ²⁺缔合和 H8 对于稳定 HwMR M 状态至关重要，HwMR M 状态是一种众所周知的光感受器信号传导状态。