Stoichiometry of proton release during photosynthetic water oxidation: a reinterpretation of the responses of Neutral red leads to a non-integer pattern

Abstract

We studied the stoichiometry of proton release into the thylakoid lumen as caused by a series of one-electron turnovers of the photosynthetic water oxidase. Earlier results obtained with Neutral red as an indicator for pH transients in the thylakoid lumen led to a 1:0:1:2 pattern of proton release for the S 0 →S 1 :S 1 →S 2 :S 2 →S 3 :S 3 →(S 4 )→S 0 transitions of the water oxidizing enzyme. Recently, a new interpretation of the Neutral red signals has been presented, that led to a non-integer stoichiometry of proton release (Lavergne, J. and Rappaport, F. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., ed.), Vol. I, pp. 873–876, Kluwer, Dordrecht). This new interpretation was based on the proposal that in stacked thylakoids the absorption changes of Neutral red reflect not only pH transients in the lumen, but also pH transients in the external partitions between the appressed membranes in grana stacks. The latter space was not accessible to the buffer bovine serum albumin which was used to quench pH transients in the bulk. This interpretation was scrutinized with both, stacked and totally unstacked membranes. In unstacked membranes, bovine serum albumin quenched pH transients at the outer side of the membrane very rapidly. Then, the pattern of proton release as detected with Neutral red revealed the proposed non-integer stoichiometry, in which a fraction of a proton (about 0.5) is released upon the S 1 →S 2 transition and less than two protons (about 1.5) during the oxygen-evolving S 3 →(S 4 )→S 0 step.