Spectrophotometric identification of the pigment associated with light-driven primary sodium translocation in Halobacterium halobium.

It has been recently proposed that in addition to bacteriorhodopsin, Halobacterium halobium membranes contain a second light-reactive pigment, whose function is the translocation of sodium ions. The evidence is based on the presence of light-driven, uncoupler-insensitive, sodium ion extrusion and light-driven, uncoupler-enhanced, passive proton uptake in cell envelope vesicles prepared from H. halobium. However, direct spectrophotometric demonstration of the existence of such a pigment had not been possible. H. halobium strain ET-15 membranes lack spectroscopically detectable quantities of red carotenoids and bacteriorhodopsin. After such membranes are extensively illuminated (bleached) in the presence of hydroxylamine, addition of trans-retinal causes the appearance of an absorption band at 588 n m , while the absorption band of free retinal decreases. The 588 nm band corresponds approximately to action spectra reported earlier for the light-driven sodium pump of H. halobium, and its magnitude in partially bleached vesicle membranes is proportional to the extent of restoration of light-induced passive proton uptake after retinal addition. Hence, we have concluded that the 588 nm absorption band is associated with the sodium pump. From the amplitude of the absorption band and the estimated extinction coefficient (48,000 M-’ cm”), the amount of the pigment in the ET-15 strain is calculated to be about 5% of the bacteriorhodopsin content of strain R-1. Although bleaching under the conditions employed, and reconstitution with retinal, are properties of bacteriorhodopsin, the 588 nm pigment is distinguished from the purple pigment by its red-shifted absorption band, by its lowered molar extinction coefficient, by its apparent lack of cis-trans isomerization (dark adaptation), by a large pH-dependent blue shift in its absorption band (from 588 to 548 nm, with a pK of 9.6), and by its sensitivity to heating at 75°C fo r 5 min, for both transport function and retinal reconstitution.