Abstract Flavodiiron proteins (FLVs) catalyze the reduction of oxygen to water by exploiting electrons from Photosystem I (PSI). In several photosynthetic organisms such as cyanobacteria, green algae, mosses and gymnosperms, FLV-dependent electron flow protects PSI from over-reduction and consequent damage especially under fluctuating light conditions. In this work we investigated biochemical and structural properties of FLVA and FLVB from the model moss Physcomitrium patens . The two proteins, expressed and purified from Escherichia coli , bind both iron and flavin cofactors and show NAD(P)H oxidase activity as well as oxygen reductase capacities. Moreover, the co-expression of both FLVA and FLVB, coupled to a tandem affinity purification procedure with two different affinity tags, enabled the isolation of the stable and catalytically active FLVA/B hetero multimer protein complex, that has never been isolated and characterized so far. The multimeric organization was shown to be stabilized by inter-subunit disulfide bonds. This investigation provides valuable new information on the biochemical properties of FLVs, with new insights into their in vivo role and regulation.

Physcomitrium patensflavodiiron proteins form a redox-dependent heterocomplex