The proton uptake channel of bacteriorhodopsin as studied by a photoelectrochemical method.
A series of the mutant proteins (D96N, D96N/D85N, D115N, L93T, T46V, V49A) where the residues are located at the cytoplasmic domain of bacteriorhodopsin (bR) were studied photoelectrochemically and their photocurrent response characteristics at the electrode/electrolyte interface were compared with those of the wild-type bR. While the wild-type bR of normal proton pumping activity yields symmetrical cathodic (positive) and anodic (negative) responses, corresponding to proton release and proton uptake, respectively, these mutants, with the exception of D115N, showed diminished amplitudes in the negative response. This indicates retardation of proton translocation from the cytoplasmic surface to the retinal Schiff base. The mutation that gave the strongest influence on the negative response was D96N while moderate influence was obtained with L93T, T46V, and V49A. These results suggest that residues other than D96 also participate in the cytoplasmic proton uptake channel, either by interacting with D96 directly or by forming a hydrogen-bonded network with water molecules. The D96N/D85N double mutant yielded little response at neutral pH, but the response was partially recovered by addition of azide, while it was fully recovered in the single mutant D96N. The D115N mutant showed the response profile that closely resembles the wild-type, indicating that D115 is not crucially involved in the event of proton transfer relay at the cytoplasmic region. It was also found that every mutant in this study releases protons prior to uptake at the other membrane surface, as does the wild-type.
Koyama
K
K
Ashigara Research Laboratories, Fuji Photo Film Co., Ltd., Minamiashigara, Kanagawa, Japan. kkoyama@ashiken.fujifilm.co.jp
Miyasaka
T
T
D001386:Azides / Q000494:pharmacology; D001436:Bacteriorhodopsins / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D010777:Photochemistry / Q000379:methods; D017354:Point Mutation; D017494:Proton Pumps / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship
D001386:Azides; D017494:Proton Pumps; D001436:Bacteriorhodopsins