Combinatorial Mutagenesis and Structural Simulations in the Environment of the Redox-Active Tyrosine YZ of Photosystem II

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Abstract
Tyr161 of the D1 protein (Yz) is a redox component closely associated with the water-oxidizing complex of photosystem II. Yz reduces the primary donor P680+, and Yzox is then rereduced by the manganese cluster that oxidizes water. We aimed to investigate whether water oxidation by P680+ could occur through an alternative pathway in the absence of Tyr161. For this purpose, combinatorial mutagenesis was performed in residues presumed to be in the environment of Tyr161. Full sequence degeneracy was introduced in two regions of the D1 protein: at codons 157, 158, 160, 162, 163, 164, and 165, which are close to Yz by sequence, and at codons 186-191 which are assumed to be close to Yz in the tertiary structure; at position 161, the nucleotide combinations were designed to not give rise to a Tyr codon. The combinatorial DNA mixture was used to transform an obligate photoheterotrophic mutant (Y161W) of the cyanobacterium Synechocystis sp. PCC 6803, in which Trp at position 161 impairs photosynthetic activity. Transformants were selected in which photoautotrophic growth was restored, resulting in 11 viable mutants. In all of these mutants, however, a Tyr codon was found at position 161, introduced either by complex repair processes or as a result of PCR-induced mutations. Additional mutations found in residues neighboring Tyr161 mostly retained photosystem II properties similar to those of wild type. However, in two of these mutants, FVEYPI and FLVYNI, photoautotrophic growth was impaired and the relative variable fluorescence was reduced. Computer simulations of the environment of Yz suggest that the position of Tyr161 varies with respect to some neighboring residues without major functional consequences. We conclude that Tyr161 fulfills a critical role through its chemical nature and positioning and that this function cannot be substituted by another residue at a nearby position.