Photovoltage evidence that Glu‐204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin

Abstract

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the pK of E204 in the M state is close to the pK of D85 in the bacteriorhodopsin ground state ( 6 6 3) and far below the pK of the terminal proton release group (V6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.z 1998 Federation of European Biochemical Societies.

Mechanism of a proton pump analyzed with computer simulations

“…One of the first questions that needed to be addressed for understanding the second proton transfer step (from the extracellular proton release group to the extracellular bulk) was the exact nature of the proton release group. Earlier experiments indicated the involvement of E204 either as the proton release group [80] or as the group that delivers the released proton to E194 [81,82]. Based on more recent data, it was proposed that the proton is stored in a cluster of water molecules that interacts with protein amino acids [83,84].…”

Opening the Schiff base moiety of bacteriorhodopsin by mutation of the four extracellular Glu side chains

“…A number of studies performed on mutated BR suggest that Glu‐194 and Glu‐204, within a hydrogen‐bonded network, form part of the proton release pathway [15–23]. However, the source of the released proton in the extracellular space, i.e.…”

Time-Resolved FT-IR Spectroscopic Investigation of the pH-Dependent Proton Transfer Reactions in the E194Q Mutant of Bacteriorhodopsin

“…Therefore, it can be concluded that E194 and E204 play a crucial role in proton release and E9 as well as E74 are not necessary for normal proton transfer to the extracellular membrane surface. Recently, several authors suggested that the released proton originates from E204 (or several groups including E204: the E204 site), whereas E194 acts as the acceptor of the proton from the E204 site and as the proton donor to the membrane surface (27,29,30). Because a band due to deprotonation of E204 (28, 31) or E194 (28) is not observed in time-resolved infrared difference spectra it was concluded that the proton is released by a hydrogen-bonded network connecting D85 and E204 and possibly including E194 and bound water.…”