A new spectral species of the purple membrane of Halobacterium halobium has been observed below pH 3.2. The formation of this new species is temperature-dependent and is favoured by increasing temperature up to the physiological range of the organism. The rate of formation at pH 3.0 and 22 degrees C is 7.9 x 10-3s-1. The spectral distribution and temperature-dependence of the new species suggest that it may be phototransiet O, stabilized by low pH. Flash-photolytic experiments in the pH range 7.2-2.7 show a pH-dependence corresponding to the static events and are consistent with a single protonation of bacteriorhodopsin below pH 3.22. These results can also be interpreted in terms of the stabilization of phototransient O at low pH. The temperature-dependence of the formation of the acid-induced species may reflect a relationship with the phase transition of the membrane.

Abstract

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Ferrocytochrome c has been shown to bind two molecules of CO at pH 14. The second CO is thought to be bound only when the cytochrome c molecule is denatured, and once bound appears to be spectrally silent. Insolubilization of native cytochrome c prevents the binding of the second CO molecule. A scheme is proposed to explain these observations based on evidence from static titrations and flash-photolysis experiments, use of carboxymethyl cytochrome c and insoluble cytochrome c, and use of cyanide instead of CO as a ligand.

The formation and reversal of the acid species of purple membrane generated below pH 4.00 (22 degrees C) is studied together with the photochemical cycle over the pH range 6.40--3.20. The buffering capacity of the membrane reaches a peak at pH 4.30, indicating the possibility of a conformational change taking place. The generation of the new spectral species can take place in the dark and is unaffected by the addition of reducing agents. Kinetic parameters measured indicate that the group being titrated below pH 4.00 could be the same as that protonated in the formation of intermediate O. The temporal placement of intermediate O after M in the photochemical cycle is shown to be incompatible with the data presented here. Reneutralization of acidified purple membrane shows that the spectral changes in acid are reversible but the phototransient properties are altered.