Chemical reconstitution of a chloride pump inactivated by a single point mutation.

Abstract

The arginine residue R108 plays an essential role in the transport mechanism of the light‐driven anion pump halorhodopsin (HR) as demonstrated by complete inactivation of chloride transport in mutant HR‐R108Q. In the presence of substrate anions, guanidinium ions bind to the mutant protein with affinities in the mM range, thereby restoring transport 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 spectra of HR wild type and HR‐R108Q‐guanidinium demonstrates that no structural changes occur in the reconstituted mutant and that the photoreactions of wild type and reconstituted mutant are identical. Furthermore, an IR absorbance band of the guanidino group of R108 can be identified at 1695/1688 cm‐1. In HR‐R108Q, a guanidinium ion binding close 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 mechanism is reconstituted.

Three-dimensional Structure of l-2-Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 Complexed with the Substrate-analogue Formate

“…This explains the importance of the serine for substrate binding affinity, as observed by Kurihara (9). In contrast, the two positively charged residues in the active site cavity, Arg 39 cally important residue contributed by the large atrium subdomain, Arg 39 , points straight into the active site. Thus, it makes a very likely candidate for halide stabilization and abstraction as it forms a positively charged cradle together with the edges of the phenyl rings of Tyr 10 and Phe…”

“…The halogen atom is held in position in a stabilizing halide cradle formed by Arg 39 , Tyr 10 , and maybe also Phe 175 . Subsequently, at the pH optimum of 9 -10, the negatively charged Asp 8 is free to attack the C-2 of the substrate to form an ester intermediate with concurrent cleavage of the carbon-halogen bond.…”

Chloride and proton transport in bacteriorhodopsin mutant D85T: different modes of ion translocation in a retinal protein 1 1Edited by A.R.Fersht

“…the guanidino group of this arginine residue, in the transport mechanism and anion binding. Moreover, addition of external guanidinium could restore anion transport in arginine-lacking mutants of halorhodopsin (Ru È diger et al, 1995). It might therefore be assumed that arginine 82, which is conserved in all halorhodopsins and bacteriorhodopsins, has this ion-binding capacity in bacteriorhodopsin as well.…”

Ligand, Cofactor, and Residue Vibrations in the Catalytic Site of Endothelial Nitric Oxide Synthase

“…The major peaks and troughs are marked on the spectra in Figure 2 and listed in Table 1. The guanidinium ν as of the substrate arginine bound to eNOS probably shows in the photolysis spectra of eNOS; in the vibration at 1691/1684 cm -1 (Figure 2A), this shifts to 1621/1602 cm -1 in D 2 O ( Figure 2B) (40,41,48). Strong differences at 1693/1684 and 1620/1604 cm -1 in the H 2 O minus D 2 O double difference spectrum further support these assignments ( Figure 2C).…”