The reaction of H2S with the photosynthetic water-oxidizing complex and its lack of reaction with the primary electron acceptor in spinach

Inhibition of photosynthetic water oxidation by H₂S, 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 NH₂OH. Like NH₂OH, H₂S inhibits formation of the S₂ oxidation state of the water oxidizing complex by a two-step process in the dark. Initially, reversible inhibition of S₂ occurs upon binding to a high affinity site in the dark (S₁) state at a low concentration of inhibitor (50% inhibition: 0.07 μmol H₂S/mg Chl, corresponding to about 15 H₂S/PS II). This causes no loss of steady-state O₂ 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 Mn²⁺/PS II using mild washing conditions, with parallel loss of O₂ evolution activity. This stoichiometry of Mn release is preserved throughout the entire concentration range of inhibition by both H₂S and NH₂OH, 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 S₂ multiline signal to a cluster of 3-4 Mn ions. The concentration of H₂S which induces 50% irreversible inhibition is 17-fold greater than that required for NH₂OH. Qualitatively, the weakerinhibition by H₂S is consistent with its lower oxidation potential compared to N₂OH. However, the quantitative agreement is poor, suggesting that other environmental factors must be involved in determining their relative inhibition strenghts. Unlike NH₂OH, H₂S 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 NH₂OH. H₂S is therfore unable to bind to the NH₂OH site on the acceptor side of PS II.