Relationships Between Proton Translocation, Absorption Changes, and Photocurrents in the Bacteriorhodopsin Photocycle

The mechanisms of proton release and uptake in bacteriorhodopsin (bR) were investigated using steady state and kinetic spectrophotometry and photocurrent methods. First, a kinetic model for the formation of the crucial M intermediate of the photocycle was formulated. The proposed model involved the refinement of previously published kinetic models by including the influence of coupling between the Schiff base counterion, D85, and the proton release group, E204. The refined model of M formation predicted that the pH dependence of the so-called B2 photocurrent of bR should reflect the titration of E204 in the photocycle. This was experimentally verified, showing the consistency of the model as well as suggesting that B2 contains a component due to proton release. Similar experiments were performed on the control mutant E204Q, in which the B2 photocurrent showed no change with pH in the appropriate range. A comprehensive interpretation of the B2 photocurrent was developed. The models of M formation and the B2 photocurrent were used to analyze the photocycles and proton release behaviour of the R82Q and R134K mutants. The pK$\sb{\rm a}$ values of D85 and E204, both in the unphotolyzed state and in the M intermediate, were measured in these mutants. The altered photocycling and proton pumping behaviour of these mutants was explained in light of the altered pK$\sb{\rm a}$s of these residues relative to the wild type protein. Proton uptake and release were investigated in the E204Q mutant. It was found that these proton transfer steps were rate limiting in the photocycle and coincided with the rise and decay, respectively, of the O intermediate. The slow decay of O was found to be due to a high activation barrier of D85 deprotonation, which could be lowered by addition of azide and other weak acids. Weak acids accelerated the decay of O in wild type bR at low pH as well. Finally, charge translocations in the 13-cis photocycle of the R82K mutant were investigated. Interestingly, these photocurrents were influenced by the same residues, namely D85 and E204, which affect the photocurrents of the all-trans photocycle.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Misra, Saurav

Contributors dc:contributor

• Ebrey, Thomas G.

• Biology, Cell

Language dc:language

eng

Identifier

(MiAaPQ)AAI9812711

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/85482