A Rapid Screening Method for Testing Modification to Photosystem I’s Reducing end by Reconstitution with Recombinantly Expressed Protein Subunits

Photosystem I (PSI) is a powerful light-induced reductase that is an appealing target for integration into hybrid solar fuel production system. Its highly reducing end is comprised of two iron-sulfur cluster redox cofactors that are assembled into PsaC, a peripheral subunit that is non-covalently attached to the transmembrane core. Two additional subunits, namely, PsaD and PsaE strap PsaC onto PSI`s membrane core, thereby creating the so-called “stromal ridge”. Unfortunately, efficient transfer of multiple electrons from PSI to potential redox catalysts is limited by diffusion of the native terminal acceptor, plant-type ferredoxin-a soluble single-electron carrier protein, to and from PSI’s reducing end. Modifying the stromal ridge to provide a faster, more direct electron transfer interface to redox catalytic centers is highly desired. But, most mutations or changes of this part are lethal to the photosynthetic organism. An alternative is chemically stripping the stromal ridge from the core complex followed by reconstitution with recombinantly expressed subunits. This approach is complicated since PsaC can only be expressed recombinantly in its apoprotein form and has to be reconstituted with iron-sulfur clusters in vitro. We are developing means of heterologously expressing PsaC as a holoprotein. This will make it possible to recombinantly co-express all stromal ridge components and rapidly test their assembly onto the core complex. Thus we will construct a rapid screening method that will enable testing modifications to the stromal ridge subunits according to our needs.