Characterization of the cation binding sites of the purple membrane. Electron spin resonance and flash photolysis studies

Mireia Dunach, Esteve Padros, Michel Seigneuret, Jean Louis Rigaud

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The binding of Mn2+ and La3+ to the blue membrane prepared by deionization of the Halo-bacterium halobium purple membrane has been studied by electron spin resonance (ESR) spectroscopy, visible absorption spectroscopy, and flash photolysis. ESR studies indicated that 10 Mn2+ binding sites are present per bacteriorhodopsin monomer. Five high- and medium-affinity sites, normally occupied by Ca2+ and Mg2+ in the purple membrane, as well as five low-affinity sites were found. Proteolysis and chemical modification experiments indicated that the low-affinity sites are located on the bacteriorhodopsin C-terminal segment, while the high- and medium-affinity sites involve other carboxyl groups of the protein. Competition experiments indicated that La3+ binds much more strongly than Mn2+ to these sites. Visible absorption spectroscopy and flash photolysis experiments indicated that binding of Mn2+ or La3+ regenerates both the purple color and formation of the M412 intermediate. The effect occurs progressively as cations bind to the high- and medium-affinity sites, bound La3+ being more effective than bound Mn2+. In addition, La3+ was also shown to inhibit the M412 decay but at concentrations higher than those required for binding to divalent cation sites. It is suggested that divalent cations support both the purple color and proton-pumping activity by rendering less negative the surface potential of the purple membrane. This process may promote deprotonation of the counterion of the retinal Schiff base and possibly of other functional groups. On the other hand, it is proposed that the inhibitory effect of La3+ is mainly due to binding to a site distinct from those of divalent cations. This latter site may be involved in Schiff base reprotonation. © 1987, American Chemical Society. All rights reserved.
Original languageEnglish
Pages (from-to)1179-1186
Issue number4
Publication statusPublished - 1 Jan 1987


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