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In an attempt to establish a relationship between proton pumping and the photocycle intermediates of bacteriorhodopsin, we have studied the effects of pH and temperature on flash-induced proton pumping and the photointermediates O640 and M412. The relative quantum yield of flash-induced proton pumping is both pH and temperature dependent. It is high in the acid pH range and at low temperatures but decreases in the basic pH range and at high temperatures. The decay of M412 is biphasic. The amplitude of the slowly decaying component (Ms) was found to be pH dependent with a pK similar to that of the delta pH. The pH dependence of the fast-decaying component (Mf) is opposite to that of Ms and delta pH. Like that of Ms, the amplitude of O640 is high in the acid pH range, but unlike the amplitude of Ms, it declines very rapidly at pHs greater than 6.5; the amplitude of O640 becomes zero around pH 8. The temperature dependence of the amplitude of Ms was found to be similar to that of delta pH, being high at low temperatures and low in the high-temperature range. The temperature dependence of Mf and O640 is opposite to that of delta pH and Ms. Whereas, O640 and Ms seem to be likely candidates (from the pH dependence) for coupling between proton pumping and the photocycle, Ms is more likely because (i) the relative quantum yield of both Ms and delta pH seem not to go to zero at higher pHs and (ii) Ms and delta pH have similar temperature dependence.

Time-resolved, flash-induced difference absorbance spectra (300-700 nm) at pH 10.5 and 5°C for the bacteriorhodopsin photocycle fast and slow decaying forms of the M intermediate (Mfand Ms, respectively) and R intermediate are reported. The main distinguishing features are as follows: For Mf, ΔAmax= 412 nm, a shoulder at 436 nm, no absorbance change at 350 nm; ΔAmin= 565 nm; ΔA412/ΔA565= 0.85. For Ms, ΔAmax= 412 nm, a shoulder at 386 nm; ΔAmin= 575 nm; ΔA412/ΔA575= 0.6. For R, ΔAmax= 336 and 350 nm (double peak), smaller peaks at 386 and 412 nm; ΔAmin= 585 nm; ΔA350/ΔA585= 0.2. The different difference spectra for Mfand Msprovide direct evidence that these species, initially identified by their kinetics, are physically distinct. With fast transient absorption spectroscopy, it was shown that R may form very fast, perhaps faster than the L intermediate decays. On the basis of the different bleaching peaks for Mfand Ms, we propose that Mfand Msare in independent photocycles formed from slightly different forms of bacteriorhodopsin. R may also be in a different photocycle. The different forms of bacteriorhodopsin are probably in dynamic equilibrium with their ratios, controlled by pH and temperature.

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 (λmax≈ 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.

Although there is good agreement that light reduces the amount of cyclic GMP (cGMP) in the retina, the exact time-course of this decrease is not well established. Bullfrog retinal sections were isolated under infrared light and quick-frozen with liquid nitrogen-cooled, metal hammers after exposure to various intensities of continuous illumination. This quick-freezing should stop the degradation of cGMP within 50-100 ms. The frozen retinal sections were then slowly warmed up in the presence of perchloric acid to denature enzymes involved in cGmp metabolism. cGMP was determined by radioimmunoassay and comparison was made between light- and dark-adapted retinal sections from the same animal. The average cGMP concentration was 44.3 +/- 0.7 pmol cGMP/mg protein or 170.9 +/- 3.2 pmol cGMP/retina. After 1 s of illumination no significant change in cGMP concentration was found even with the brightest light used (approximately 7 x 10(7) rhodopsins bleached/second per rod. At this intensity the first significant decrease in cGMP from dark-adapted levels was detected 3-5 s after the initiation of illumination; cGMP decayed to 70-75% of the dark-adapted value after approximately 30 s. With lower intensity illumination the cGMP levels recovered to dark-adapted levels after the initial decrease even though the bleaching light remained on.