title: Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin.
abstract: Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.
authors.affiliation: School of Chemical Sciences, University of Georgia, Athens 30602.
authors.forename: R C
authors.initials: RC
authors.lastname: Conover
authors.forename: A T
authors.initials: AT
authors.lastname: Kowal
authors.forename: W G
authors.initials: WG
authors.lastname: Fu
authors.forename: J B
authors.initials: JB
authors.lastname: Park
authors.forename: S
authors.initials: S
authors.lastname: Aono
authors.forename: M W
authors.initials: MW
authors.lastname: Adams
authors.forename: M K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D001105:Archaea / Q000378:metabolism*; D001419:Bacteria / Q000378:metabolism*; D002942:Circular Dichroism; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000302:isolation & purification / Q000378:metabolism; D007501:Iron / Q000032:analysis; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D013455:Sulfur / Q000032:analysis; D013816:Thermodynamics
chemical_list: D005288:Ferredoxins; D004227:Dithionite; D013455:Sulfur; D007501:Iron