Expression, purification and characterization of an active C491G variant of ferredoxin sulfite reductase from Synechococcus elongatus PCC 7942

Recently developed molecular wire technology takes advantage of [4Fe-4S] clusters that are ligated by at least one surface exposed Cys residue. Mutagenesis of this Cys residue to a Gly opens an exchangeable coordination site to a comer iron atom that can be chemically rescued by an external thiolate ligand. This ligand can be subsequently displaced by mass action using a dithiol molecular wire to tether two redox active proteins. We intend to apply this technique to tethering Photosystem Ito ferredoxin sulfite reductase (FdSiR), an enzyme that catalyzes the six-electron reduction of sulfite to hydrogen sulfite and nitrite to ammonia. The enzyme contains a [4Fe-4S](2+1+) cluster and a siroheme active site. FdSiR(WT) and an FdSiR(C491G) variant were cloned from Synechococcus elongatus PCC 7942 and expressed along with the cysG gene from Salmonella typhimuriwn using the pCDFDuet plasmid. UV/Vis absorbance spectra of both FdSiR(WT) and the FdSiR(C491G) variant displayed characteristic peaks at 278, 392 (Soret), 585 (alpha) and 714 nm (charge transfer band), and 278, 394 (Soret), 587 (alpha) and 714 nm (charge transfer band) respectively. Both enzymes in their as-isolated forms displayed an EPR spectrum characteristic of an S = 5/2 high spin heme. When reduced, both enzymes exhibited the signal of a low spin S = 1/2 [4Fe-4S](1+) cluster. The FdSiR(WT) and FdSiR(C491G) variant both showed activity using reduced methyl viologen and Synechococcus elongatus PCC 7942 ferredoxin 1 (Fd1) as electron donors. Based on these results, the FdSIR(C491G) variant should be a suitable candidate for wiring to Photosystem I.