A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal FB [4Fe-4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaCC14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe-4S]1+ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of FA-, and the minority set had g values of 2.11, 1.90, and 1.88 from FB' in the modified site. The S = 1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe-4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe-4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaCC14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe-4S] and [4Fe-4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.
Keywords: Cyanobacteria; Electron transfer; Iron–sulfur cluster; Photosynthesis; Photosystem I; PsaC.