Using targeted mutagenesis to investigate the role of the bicarbonate ligated to the non-heme iron of Photosystem II

Abstract

Photosystem II (PS II) is a photochemical oxidoreductase enzyme capable of splitting water and initiating photosynthesis. Photosystem II functions by removing electrons from water, and transferring them via a series of cofactors to a bound quinone QA. The electron is then passed to an exchangeable plastoquinone QB, which after a second reduction can dissociate as reduced plastoquinol, to be replaced by a new oxidized plastoquinone. Located between QA and QB is a bicarbonate molecule ligated to a non-heme iron (NHI). This bicarbonate appears to participate in a hydrogen bonding network which connects the NHI with the cytosol (or stroma in plants). In addition to the bicarbonate, the hydrogen bonding network is made up of many residues from the DE loop of the D1 subunit of PS II. The purpose of this study was to investigate the role that the bicarbonate ligand plays in PS II function. To achieve this, targeted mutagenesis was used to generate a series of mutants within the bicarbonate environment in the cyanobacterium Synechocystis sp. PCC 6803. Additionally, mutants were made in the DE loop of the D1 subunit, to investigate how the DE loop affects the bicarbonate environment. Physiological characterisation of the mutant strains indicated that the bicarbonate ligated to the NHI plays a role in QA- to QB electron transfer. Additionally, following migration of the electron onto QB, the participation of the bicarbonate in the hydrogen bonding network is required for rapid protonation of reduced QB- or QB2-(H+). Furthermore, the alteration of the bicarbonate environment frequently resulted in sensitivity to high light, supporting the putative role of bicarbonate in limiting the production of reactive oxygen species during high light conditions. Finally, the mutation of the DE loop indicated that restriction of DE loop movement is important for effective PS II activity. The characterisation of the DE loop mutants also led to the proposal of a model for the process of PS II repair following photodamage, whereby, photodamage to the DE loop induces DE loop flexibility, which initiates the recycling of damaged PS II protein.