A research poster presented at GRC Photosynthesis 2023, Maine, USA. Abstract This project focuses on the evolutionary engineering of Photosystem II (PS II), a crucial enzyme in photosynthesis that powers all life on Earth by harvesting electrons and protons from water. Despite being one of the slowest evolving enzymes and well-conserved across phyla, PS II is an ever-evolving entity (1). Directed evolution efforts have been made to explore its evolvability beyond its current arrangement, using an error-prone PCR based in vitro reaction centre subunit D1 library creation platform with D1 deficient Synechocystis sp. PCC 6803 strain. Resultant mutant libraries with variant PS II were tested for atypical light conditions, herbicides, and a possible alternative transition metal cluster by substituting manganese with ruthenium. Several better-performing clones are characterised by biochemical analyses and used as primers for the next round of evolution. Recent advances in machine learning-based protein structure prediction have also been utilised (AlphaFold 2) to predict the structures of over 700 available D1 sequences spanning cyanobacteria to higher plants, as well as ancestral D1 and D0 (2). These structures were then fed into mTM-align (3) for global and pairwise structural alignments, consensus backbone and distance matrix tree, and residue-level conservation scores were obtained through ConSurf analysis (4). Insights gained from this analysis shed light on the structural divergence of PS II and potential targets for more informed mutagenesis. Comparison with the traditional multiple sequence alignment approach provided further information about known residues versus structural changes. Together, these results provide a promising direction for further exploration of PS II evolvability beyond water oxidation. Reference 1. T. Oliver et al., The Evolution and Evolvability of Photosystem II. Annu Rev Plant Biol 74, (2023). 2. T. Oliver, P. Sanchez-Baracaldo, A. W. Larkum, A. W. Rutherford, T. Cardona, Time-resolved comparative molecular evolution of oxygenic photosynthesis. Biochim Biophys Acta Bioenerg 1862, 148400 (2021). 3. R. Dong, Z. Peng, Y. Zhang, J. Yang, mTM-align: an algorithm for fast and accurate multiple protein structure alignment. Bioinformatics 34, 1719-1725 (2018). 4. B. Yariv et al., Using evolutionary data to make sense of macromolecules with a "face-lifted" ConSurf. Protein Sci 32, e4582 (2023).