Buffer Effect on the Photoelectrochemical Response of Bacteriorhodopsin

Abstract

Bacteriorhodopsin (bR) is the retinal protein functioning as a light-driven proton pump in Halobacterium salinarium. Photoisomerization of the retinal chromophore triggers the proton transport from the cytoplasmic side to the extracellular side of the membrane, and the electrochemical potential thus built up across the membrane is then used for ATP synthesis. 1 The proton transport is a cyclic process consisting of a series of intermediates, with lifetimes varying from picoseconds to milliseconds. 2,3 bR could be a promising material for use in bioelectronics, because of its outstanding thermal, chemical, and photochemical stability. In view of this, the photoelectrochemistry of bR has attracted much attention recently.4-12 bR on a tin oxide (SnO 2 ) electrode generates, by turning the incident light on and off, transient photocurrents, hereafter called the light-on and light-off photocurrents, respectively. This unique photoresponse could be applied to optical sensors and a bR-based artificial photoreseptor. 4,5 The origin of the transient photocurrent from bR at electrode-electrolyte interfaces, however, still remains ambiguous.We showed, by use of a gold electrode, that the photocurrent intensity well paralleled the amount of the surface oxide. This result suggests that the transient photocurrent would originate in the interfacial pH change via proton release/uptake by bR. 11Several works have so far dealt with the pH dependence of the bR photoresponse. The photocurrent polarity reversal at low pH 8,9,12 and the light-off photocurrent intensity loss at higher pHs 12 , were correlated with the proton release/uptake sequence in the bR photocycle. The pH profile of the photocurrent intensity, however, was not always compatible with the proton pump activity 13 of bR. Further, though pH buffers were used in some cases, their effects have not been elucidated in detail.In the present work, we have closely examined the effect of buffer agents on the bR photoresponse to find a systematic correlation between them for the first time. Materials and MethodsPurple membranes were prepared from cultured H. salinarium S9 according to the method of Oesterhelt and Stoeckenius 14 and were suspended in pure water until use.SnO 2 electrodes (Nippon Sheet Glass Co., with a 450-nm thick SnO 2 layer) were washed first with hot methanol, then treated with hot concentrated sulfuric acid, and rinsed thoroughly with distilled water. A 50-µl aliquot of the bR suspension, with an optical density of 1 at 570 nm for 1-cm pathlength, was deposited on a 1-cm 2 area of the electrode, and then dried at room temperature and humidity to obtain a bR-immobilized electrode.The photocurrent measurement setup was essentially the same as the one reported eleswhere.11 An Ushio Electric 500-W xenon arc lamp Model UXL-500D-O served as the light source. Infrared radiation was removed with a 18-cm pathlength water cell, and Toshiba Glass cut-off filters L-39 and G-55S were used to pass green light. A bR-immobilized electrode was mounted as a window (1 cm in ...

A Tin Oxide Transparent Electrode Provides the Means for Rapid Time‐resolved pH Measurements: Application to Photoinduced Proton Transfer of Bacteriorhodopsin and Proteorhodopsin†

“…2A,B. A similar observation was reported previously (23). In addition, when BR, a light‐driven H + pump, adsorbed on the electrode surface was replaced with halorhodopsin, a light‐driven Cl − pump, no light‐induced signals were observed.…”

Efficient Approach to Determine the pKa of the Proton Release Complex in the Photocycle of Retinal Proteins

“…A gradual magnitude decrease was observed in high buffer capacity as in the previous work . The decrease depends on buffer capacity and indicates that the electric signals are caused by pH change, which can be almost completely eliminated by adding a large amount of buffer, for instance, >50 mM for a thin AR4 film as shown in Figure b.…”

“…A buffer can affect the amplitude and decay time of photoelectric signals. ,,− It was shown with pH-sensitive dyes that buffers accelerate release of a proton from a membrane surface to the bulk . The present studies examine buffer effects via the photoelcterochemical approach.…”

Buffer Effects on Electric Signals of Light-Excited Bacteriorhodopsin

“…To explain the buffer effect associated with the B2 component Liu et al assumed that the protons emitted into the solution during M formation protonated the buffer molecules, which were repelled or attracted ("positive" buffers or "negative" buffers, respectively) by the negative charge remaining on the membrane surface after proton release (Liu et al, 1991). Recently, Saga et al (1999) reported on the effect of buffers on the photoelectrochemical response of bR.…”