The reaction of H 2S with the photosynthetic water-oxidizing complex and its lack of reaction with the primary electron acceptor in spinach

Abstract

Inhibition of photosynthetic water oxidation by H 2S, a substrate analog, has been investigated using equilibrium titrations and EPR spectroscopic detection of the electron donors in spinach Photosystem II (PS II) membranes and compared to inhibition by NH 2OH. Like NH 2OH, H 2S inhibits formation of the S 2 oxidation state of the water oxidizing complex by a two-step process in the dark. Initially, reversible inhibition of S 2 occurs upon binding to a high affinity site in the dark (S 1) state at a low concentration of inhibitor (50% inhibition: 0.07 μmol H 2S/mg Chl, corresponding to about 15 H 2S/PS II). This causes no loss of steady-state O 2 evolution and can be reversed by illumination at room temperature, which causes multiple turnovers. At higher concentrations, irreversible inhibition occurs due to the cooperative release of 3 out of 4 Mn 2+/PS II using mild washing conditions, with parallel loss of O 2 evolution activity. This stoichiometry of Mn release is preserved throughout the entire concentration range of inhibition by both H 2S and NH 2OH, suggesting a common binding site for at least 3, and possibly all 4, of the Mn ions which are present in PS II. This consistent with independent earlier work showing the net release of 4 Mn/PS II using stronger washing conditions, and also with EPR spectroscopic evidence assigning the S 2 multiline signal to a cluster of 3–4 Mn ions. The concentration of H 2S which induces 50% irreversible inhibition is 17-fold greater than that required for NH 2OH. Qualitatively, the weakerinhibition by H 2S is consistent with its lower oxidation potential compared to N 2OH. However, the quantitative agreement is poor, suggesting that other environmental factors must be involved in determining their relative inhibition strenghts. Unlike NH 2OH, H 2S does not affect the structure of the primary quinone electron acceptor, Q A −-His-Fe (structure by analogy to bacterial reaction centers), as seen by formation of the normal EPR signals at g = 1.8 and g = 1.9 for the photoreduced semiquinone, instead of shifted resonance at g = 2.1 observed with NH 2OH. H 2S is therfore unable to bind to the NH 2OH site on the acceptor side of PS II.