# Important amino acid residues in the chloride pump halorhodopsin that accelerate ion transport despite no direct interaction with the substrate

## TL;DR

This study identifies key amino acid residues in a chloride pump protein that help accelerate ion transport even though they don't directly interact with the ion.

## Contribution

The study reveals how specific hydrophobic residues in halorhodopsin indirectly influence Cl- transport by enabling conformational changes.

## Key findings

- Mutations in Phe211 and Leu214 slow Cl- transport by disrupting the positioning of Lys215.
- Hydrophobic residues are critical for forming a hydrated channel that allows efficient Cl- conduction.
- Long-lived intermediates accumulate when these residues are mutated, obstructing ion passage.

## Abstract

Ion-pump rhodopsins are widely distributed photoactive membrane proteins found in microorganisms. Their cytoplasmic (CP) regions are predominantly hydrophobic, inherently restricting substrate ion permeation. However, these rhodopsins can rapidly transport substrate ions via photo-induced conformational changes. The well-characterized H+-pumping rhodopsins employ dissociable residues such as Asp, Glu, or Lys to mediate rapid H+ relay reactions along a transiently hydrated CP pathway. In contrast, the corresponding mechanisms in other ion pumps remain poorly understood. Here, we investigated the key factors contributing to ion transport by halorhodopsin (HR), a Cl- pump from the archaeon Natronomonas pharaonis (NpHR). Upon photoactivation, NpHR creates a hydrated Cl- transport pathway in its CP region, which is surrounded by bulky hydrophobic residues that do not directly interact with Cl-. However, mutations in specific hydrophobic residues significantly slow Cl- transport. Notably, Phe211 and Leu214, located near the pathway exit, play critical roles. Mutations in these residues likely disrupt the proper positioning of the Lys215 sidechain, which inadvertently binds Cl- from the surrounding solution and positions it in a way that obstructs Cl- transport. As a result, ion passage is hindered, leading to the accumulation of long-lived intermediates. These findings suggest that the hydrophobic residues surrounding the pathway are not merely structural components. Instead, they are critical for enabling specific conformational changes that facilitate the formation of a hydrated channel, allowing efficient Cl- conduction without obstruction.