Photosystem II (PSII) is the only enzyme in nature capable of catalysing the oxidation of water.
Despite its ubiquity in plants, algae, and cyanobacteria, water oxidation has only evolved once,
in an ancestor to the cyanobacterial phylum. How and when this occurred is unknown, but many
hypotheses exist, with no clear consensus. Here, the evolution of PSII, and its bioenergetics, were
investigated using a bioinformatic and biophysical approach.
The heterodimeric reaction centre in PSII is hypothesised to have evolved froma homodimeric
ancestor, which was capable of charge separation on both sides of the reaction centre. Ancestral
Sequence Reconstruction (ASR) has been performed to predict the protein sequences of the homodimeric
and heterodimeric ancestors in PSII, named AncD0 and AncD1. Analysis of AncD0
sequences was used to rationalise charge separation, quinone chemistry, and electron donation
within the homodimeric reaction centre. Additionally, AncD1 sequences were shown to support
water oxidation, suggesting that the rogue and super-rogue D1 isoforms have lost their water
oxidation capability.
Site directed mutagenesis of the D2 subunit in PSII was used to reconstruct ancestral traits
and explain the origin of the functional asymmetry in the homodimeric RC. In a knockout of
the redox-active Tyrosine D, D2-Y160F, the S1 state of the Mn4CaO5 cluster was observed to
decay to the S0 state during long dark adaptations, in thylakoids membranes lacking the extrinsic
PSII subunits. This was not observed in intact D2-Y160F thylakoids, providing a rational for the
oxidation of S0 to S1 by Tyrosine D, and its strict conservation in all known D2 proteins. EPR
and time-resolved fluorescence measurements were used to show that the D2-R294N mutation
inhibits the formation of the stable Tyrosine D radical. It is proposed that D2-R294 evolved to
fulfil two roles: firstly, as a controller of the hydrogen-bonding direction around Tyrosine D, and
secondly, as a fine-tuner of the PD1 and PD2 redox potentials so that the cation is localised on the
D1 side of the RC.
Finally, sequence analysis and transient absorption spectroscopy was used to investigate the
function of the atypical D1 isoform, rogue D1 (rD1). The results of these analyses were combined
to propose a role for rD1-PSII as a light-activated signalling complex, used by nitrogen-fixing
cyanobacteria to indicate that nitrogen fixation should be switched off.