Halobacterium salinarum is an extremely halophilic marine Gram-negative obligate aerobic archaeon. Despite its name, this is not a bacterium, but rather a member of the domain Archaea, which lives in hypersaline lakes. Bacteriorhodopsin (BRh) is the red retinal-containing protein found in the cell membranes of H. salinarum and is considered a light-activated proton pump that transports protons across the plasma membrane. Bacteriorhodopsin photointermediates have been defined in kinetic and spectroscopic terms as BR568, K590, L550, M412, N560, and O640. We have previously shown, using the Forster cycle for BRh that its acidity increases greatly on illumination. Therefore, protons released upon illumination of the L550 intermediate with 412 nm light may not play an essential role in ATP production. Instead, the light-induced excitation energy, which represents the energy difference between the L550 and M412 states, can be used to extract an ATP molecule attached to ATP synthase. Thus, we have shown that this amount of energy corresponds to a near-infrared vibration, which is sufficient for ATP production and provides the most feasible molecular mechanism for this phenomenon. Here, we provide new evidence that protons are released due to BRh excitation, unrelated to ATP synthesis, being only a secondary phenomenon. In addition, once released from H. salinarum cells, protons should return back into the cells via ATP-synthase molecules to produce ATP. This is not possible at pH > 7.0, such as pH 9.5. However, the stability of M intermediates and ATP formation appear to be increased at higher pH values. Indeed, a spectral shift of 138 nm may be associated with an energy amount of about 17 kcal mol-1, which is enough energy to release a mole of ATP from ATP-synthase. In general, light excitation of fluorescent molecules is a phenomenon that induces a strong increase in their acidity. Recent data suggest that the chemiosmotic hypothesis put forward by Peter Mitchell to explain ATP formation in living cells is not correct, at least in terms of explaining light-induced ATP production in H. salinarum cells.
