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Reversible inhibition of the proton pump bacteriorhodopsin by modification of tyrosine 64
Abstract:
Five mol of lysine per mol of bacteriorhodopsin were modified with methylacetimidate. This treatment did not inactivate bacteriorhodopsin but prevented all lysines from subsequent reaction with diazotized sulfanilic acid. This reaction predominantly modified tyrosine 64 and light-induced proton translocation was abolished. Reduction of the mono(p-azobenzene sulfonic acid) tyrosine 64 to the corresponding 3-amino derivative with sodium dithionite led to complete reactivation of the proton translocation activity of bacteriorhodopsin. The relative location of tyrosines 26 and 64 and the COOH terminus on the two surfaces of the purple membrane was determined by incorporation into phospholipid vesicles, subsequent modification, and proteolytic treatment. The results obtained support the models proposed by Engelmann et al. (Engelman, D. M., Henderson, R. McLauchlan, A. D., and Wallace, B. A. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2023-2027) and by Ovchinnikov et al. (Ovchinnikov, Yu. A., Abdulaev, N. G., Feigina M. Yu., Kiselev A. V., and Lobanov, N. A. (1979) FEBS Lett. 100, 219-224). Tyrosine 64 is located on the extracellular side of the membrane, whereas tyrosine 26 and the COOH terminus are located on the cytoplasmic side. Because specific nitration of tyrosine 26 also leads to inactivation of bacteriorhodopsin (Lemke, H. D., and Oesterhelt, D. (1981) Eur. J. Biochem. 115, 595-604), the results obtained demonstrate that amino acid residues located on both surfaces of the purple membrane are involved in proton translocation.
Insights
Bacteriorhodopsin proton translocation involves amino acids on both membrane surfaces. Modifying tyrosine 64 inactivated the protein, but reduction restored activity, confirming its crucial role.
Area of Science:
- Biochemistry
- Membrane Biology
- Protein Function
Background:
- Bacteriorhodopsin is a light-driven proton pump crucial for cellular energy.
- Understanding the roles of specific amino acid residues in proton translocation is key to elucidating its mechanism.
Purpose of the Study:
- To investigate the role of tyrosine 64 in bacteriorhodopsin's proton translocation activity.
- To determine the membrane surface localization of key amino acid residues, including tyrosine 26 and tyrosine 64.
Main Methods:
- Chemical modification of lysine residues using methylacetimidate.
- Diazotized sulfanilic acid labeling to modify tyrosine residues.
- Spectrophotometric analysis of proton translocation activity.
- Incorporation into phospholipid vesicles followed by proteolytic treatment to determine residue localization.
Main Results:
- Modification of lysine residues did not abolish bacteriorhodopsin activity but prevented subsequent tyrosine modification.
- Diazotized sulfanilic acid predominantly modified tyrosine 64, leading to complete loss of proton translocation.
- Reduction of the modified tyrosine 64 restored proton translocation activity.
- Tyrosine 64 is located on the extracellular side, while tyrosine 26 and the C-terminus are on the cytoplasmic side.
Conclusions:
- Tyrosine 64 plays a critical role in light-induced proton translocation in bacteriorhodopsin.
- Amino acid residues on both the extracellular and cytoplasmic sides of the purple membrane are essential for proton translocation.
- The findings support existing models of bacteriorhodopsin structure and function.