No abstract

We have found that extensively washed purple membrane has about 1 calcium and 3-4 magnesium ions bound per bacteriorhodopsin molecule. When these divalent cations are removed by any of a variety of means, the pigment changes its color from purple to blue (Xmax 600 nm). This blue pigment, which can be formed at near neutral pH, is probably very similar to blue species formed when the pH of a purple membrane sample is lowered to -2. When any of a wide variety of cations are added to a blue membrane preparation, the characteristic purple color of bacteriorhodopsin returns. Divalent and trivalent cations are much more efficient than monovalent cations in restoring the purple color and are effective at a ratio approaching one cation per pigment molecule. Besides shifting the absorption spectrum, removal of the divalent cations drastically alters the photochemical cycle of bacteriorhodopsin, including abolishing the unprotonated Schiff base (M-type) intermediate. Finally, lanthanum not only displaces the divalent cations normally bound to the purple membrane but also greatly reduces both the rate of decay of the M412 intermediate and proton uptake.Bacteriorhodopsin is the only protein in the light-energytransducing purple membrane of Halobacterium halobium. Light, absorbed by the retinal chromophore of bacteriorhodopsin, leads to the creation of a metastable high-free-energy primary photoproduct, which is then used to power the transport of two or more protons across the purple membrane. The proton gradient that results can be used for the synthesis of ATP and driving other cellular processes (see reviews in refs. 1 and 2).Bacteriorhodopsin normally is purple (Xmax = 558 nm), hence the name of the purple membrane. In their original report on bacteriorhodopsin, Oesterhelt and Stoeckenius (3) also noted that at very low pH the purple membrane turned blue (Xmaxc = 603 nm), and there have been several studies of this "acid" blue membrane (4-6). Subsequently we have found that extensive washing of the purple membrane with distilled water (pH 6) often causes it to turn blue, but initially we had a great deal of trouble finding conditions that could lead to a reproducible preparation. In investigating the formation of blue membrane by washing with distilled water, we found that pretreatment with high concentrations of NaCl was essential.Here we present evidence that the transition from the purple membrane to blue membrane is due to the removal of at least one and perhaps several divalent cations, probably calcium and magnesium. These divalent cations are normally bound to bacteriorhodopsin with high affinity and play important roles in determining not only color but photochemistry. We also have found that the displacement of the Ca2+/Mg2+ with lanthanum retains the purple color of the membrane but slows down the rate of decay of the shortwavelength photocycle intermediate, M, and the rate of proton uptake. A preliminary version of these results was presented at a meeting of the Biophysical