Effects of Ethylene Glycol and Methanol on Ammonia-Induced Structural Changes of the Oxygen-Evolving Complex in Photosystem II

Abstract

Ammonia is an inhibitor of water oxidation and a structural analogue for substrate water, making it a valuable probe for the structural properties of the possible substrate-binding site on the oxygen-evolving complex (OEC) in photosystem II (PSII). By using the NH₃-induced upshift of the 1365 cm^-1 IR mode in the S₂Q_A^-/S₁Q_A spectrum and the NH₃-modified S₂ state EPR signals of PSII as spectral probes, we found that ethylene glycol has clear effects on the binding properties of the NH₃-specific site on the OEC. Our results show that in PSII samples containing 30% (v/v) ethylene glycol, the affinity of the NH₃-specific binding site on the OEC is estimated to be more than 10 times lower than that in PSII samples containing 0.4 M sucrose. In addition, our results show that the NH₃-induced upshift of the 1365 cm^-1 IR mode in the S₂Q_A^-/S₁Q_A spectrum is dependent on the concentration of ethylene glycol, but not dependent on the concentration of sucrose (up to 1.5 M) or methanol (up to 5.4 M). By comparing the concentration dependence of sucrose and ethylene glycol on NH₃-induced spectral change and also by comparing the sucrose and ethylene glycol data at similar concentrations (∼1 M), we conclude that ethylene glycol has a clear effect on the NH₃-induced spectral changes. Furthermore, our results also show that ethylene glycol alters the steric requirement of the amine effect on the upshift of the 1365 cm^-1 mode in the S₂Q_A^-/S₁Q_A spectrum. In PSII samples containing 30% (v/v) ethylene glycol, only NH₃, not other bulkier amines (e.g., Tris, AEPD, and CH₃NH₂), has a clear effect on the upshift of the 1365 cm^-1 mode in the S₂Q_A^-/S₁Q_A spectrum; in contrast, in PSII samples containing 0.4 M sucrose, both NH₃ and CH₃NH₂ have a clear effect. On the basis of the results mentioned above, we propose that ethylene glycol acts directly or indirectly to decrease the affinity or limit the accessibility of NH₃ and CH₃NH₂ to the NH₃-specific binding site on the OEC in PSII. Finally, we also applied the same approach to test whether methanol is able to compete with ammonia on its binding site on the OEC. We found that 4% (v/v) methanol does not have any significant effect on the NH₃-induced upshift of the 1365 cm^-1 mode in the S₂Q_A^-/S₁Q_A spectrum and the NH₃-modified S₂ state g = 2 multiline EPR signal. Our results suggest that methanol is unable to compete with NH₃ upon binding to the Mn site of the OEC that gives rise to the altered S₂ state g = 2 multiline EPR signal.