Threonine-89 participates in the active site of bacteriorhodopsin: evidence for a role in color regulation and Schiff base proton transfer.
Bacteriorhodopsin (bR) functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium. A major feature of bR is the existence of an active site which includes a retinylidene Schiff base and amino acid residues Asp-85, Asp-212, and Arg-82. This active site participates in proton transfers and regulates the visible absorption of bacteriorhodopsin and its photointermediates. In this work we find evidence that Thr-89 also participates in this active site. The substitution Thr-89 --> Asn (T89N) results in changes in the properties of the all-trans retinylidene chromophore of light-adapted bR including a redshift of the visible lambda(max) and a downshift in C=N and C=C stretch frequencies. Changes are also found in the M and N intermediates of the T89N photocycle including shifts in lambda(max), a downshift of the Asp-85 carboxylic acid C=O stretch frequency by 10 cm(-1), and a 3-5-fold decrease in the rate of formation of the M intermediate. In contrast, the properties of the 13-cis retinylidene chromophore of dark-adapted T89N as well as the K and L intermediates of the T89N photocycle are similar to the wild-type bacteriorhodopsin. These results are consistent with an interaction of the hydroxyl group of Thr-89 with the protonated Schiff base of light-adapted bR and possibly the N intermediate but not the 13-cis chromophore of dark-adapted bR or the K and L intermediates. Thr-89 also appears to influence the rate of Schiff base proton transfer to Asp-85 during formation of the M intermediate, possibly through an interaction with Asp-85. In contrast, the hydroxyl group of Thr-89 is not obligatory for proton transfer from Asp-96 to the Schiff base during formation of the N intermediate.
Russell
T S
TS
Physics Department, Boston University, Massachusetts 02215, USA.
Coleman
M
M
Rath
P
P
Nilsson
A
A
Rothschild
K J
KJ
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D006217:Halobacterium / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D017494:Proton Pumps; D012545:Schiff Bases / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D013912:Threonine
D017494:Proton Pumps; D012545:Schiff Bases; D013912:Threonine; D001436:Bacteriorhodopsins