CHEMICAL RECONSTITUTION OF A CHLORIDE PUMP INACTIVATED BY A SINGLE-POINT MUTATION

The arginine residue R108 plays an essential role in the transport mec hanism of the light-driven anion pump halorhodopsin (HR) as demonstrat ed by complete inactivation of chloride transport in mutant HR-R108Q. In the presence of substrate anions, guanidinium ions bind to the muta nt protein with affinities in the mM range, thereby restoring transpor t activity and photochemical properties of wild type. One guanidinium ion and one anion are bound per molecule of HR-R108Q. For HR wild type , HR-R108Q-guanidinium and HR-R108K, differences in transport activity and anion selectivity are found which may be explained by effects of anion solvation. The agreement between light-induced FTIR difference s pectra of HR wild type and HR-R108Q - guanidinium demonstrates that no structural changes occur in the reconstituted mutant and that the pho toreactions of wild type and reconstituted mutant are identical. Furth ermore, an IR absorbance band of the guanidino group of R108 can be id entified at 1695/1688 cm(-1). In HR-R108Q, a guanidinium ion binding c lose to the mutated residue is proposed to mimick the role of the R108 side chain as the anion uptake site. Thus the wild type reaction mech anism is reconstituted.