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

Karim A. Walters, John H. Golbeck

Research output: Contribution to journalArticle

Abstract

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 corner 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 I to 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. FdSiRWT and an FdSiRC491G variant were cloned from Synechococcus elongatus PCC 7942 and expressed along with the cysG gene from Salmonella typhimurium using the pCDFDuet plasmid. UV/Vis absorbance spectra of both FdSiRWT and the FdSiRC491G variant displayed characteristic peaks at 278, 392 (Soret), 585 (α) and 714 nm (charge transfer band), and 278, 394 (Soret), 587 (α) 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 FdSiRWT and FdSiRC491G variant both showed activity using reduced methyl viologen and Synechococcus elongatus PCC 7942 ferredoxin 1 (Fd1) as electron donors. Based on these results, the FdSIRC491G variant should be a suitable candidate for wiring to Photosystem I.

Original languageEnglish (US)
Pages (from-to)1096-1107
Number of pages12
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1859
Issue number10
DOIs
StatePublished - Oct 2018

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All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

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