Richard H. Lozier

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane.

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.

TESTING KINETIC MODELS FOR THE BACTERIORHODOPSIN PHOTOCYCLE—II. INCLUSION OF AN O TO M BACKREACTION

Abstract Using previously established procedures we have tested a kinetic model of bacteriorhodopsin involving a backreaction from O to M. This model is superior to the previously tested models involving only unidirectional reaction paths. However, the spectrum of O is still too strongly temperature dependent in the red. Therefore, at least one additional feature is necessary to obtain a completely consistent kinetic model of the bacteriorhodopsin photocycle.

Effect of photoselection upon saturation and the dichroic ratio in flash experiments upon effectively immobilized systems

Abstract— Flash spectroscopy of photochemical or photobiological systems, such as bacteriorhodopsin in purple membrane, for which the chromophore transition dipole moment does not undergo complete reorientation during the time of the flash, is considered as a function of light intensity. Due to photoselection, saturation proceeds very slowly with increasing flash intensity and the linear dichroic ratio decays rapidly from a maximum of three at zero flash intensity. Simple formulae are derived to describe these effects under stringent assumptions. Calculations are also performed which relax the assumptions by taking into account (i) non‐zero optical density, (ii) thermal decay of the photoproduct during the flash and (iii) non‐zero angle y between the initial chromophore and its photoproduct. Agreement with experiments on bacteriorhodopsin in purple membranes is excellent.

Photoreactions of bacteriorhodopsin

Bacteriorhodopsin is a membrane-bound light energy transducer which generates an electrochemical proton gradient. It undergoes a cyclic photoreaction in which five intermediates have been identified. During the cycle it releases a proton from one surface of the membrane and takes up a proton on the opposite surface. The active chromophore consists of retinal bound through a Schiff base to the protein. The Schiff base is deprotonized during the photoreaction cycle and appears to be involved in the transport of protons through the membrane. The retinal may also undergo an isomerization.