Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q.

Abstract:

:The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).

journal_name

Biophys J

journal_title

Biophysical journal

authors

Lazarova T,Sanz C,Querol E,Padrós E

doi

10.1016/S0006-3495(00)76749-X

subject

Has Abstract

pub_date

2000-04-01 00:00:00

pages

2022-30

issue

4

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(00)76749-X

journal_volume

78

pub_type

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