Site-directed Mutagenesis of Thermosynechococcus elongatus Photosystem II: the O₂ Evolving Enzyme Lacking the Redox Active Tyrosine D

Abstract

Site-directed mutagenesis in the photosystem II (PSII) was performed using the thermophilic cyanobacterium Thermosynechococcus elongatus. PSII lacking the redox active tyrosine Tyr_D was engineered by substituting phenylalanine for tyrosine at D2-160. The effects of the Y160F mutation were characterized. The t_1/2 of oxygen release on the S₃→S₀ transition was estimated to be 1-2 ms in both WT’ and Y160F, although the doubling time of Y160F cells was approximately twice as slow as that of WT’ cells. Flash-induced absorption changes around 430 nm revealed the displacement of the P_D1⁺P_D2 ↔P_D1P_D2⁺ equilibrium in favor of the left side. The FTIR difference spectrum of P₆₈₀⁺/P₆₈₀ showed that the carbomethoxy C=O stretch of chlorophyll downshifted about 10 cm^-1 upon mutation. The rate of P₆₈₀⁺ reduction by Tyr_Z in the mutant slightly increased. These results suggest that minor structural perturbations perhaps in the H-bonding network between Tyr_D and P₆₈₀ take place upon Y160F mutation.