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Halorhodopsin pumps Cl– and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions

Significance Changing a few residues can change the function of homologous proteins. The chloride and proton affinity in the inward chloride-pumping halorhodopsin (HR) and outward proton-pumping bacteriorhodopsin (BR) are compared using classical electrostatic simulations. BR binds and releases protons from acidic residues that have been removed from HR. In the states where these acids are ionized in BR, HR binds a chloride. In the states where these acids bind a proton in BR, HR releases the chloride. Thus, BR uses static anions and mobile protons, whereas HR uses mobile ions to maintain the same charge states. The use of mobile ions makes HR more sensitive to external conditions. Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl– is bound to HR in a position near the deleted acid. In the HR ground state, Cl– binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl– from the central T111 site and the tight binding of Cl– to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.