title: The small iron-sulfur protein from the ORP operon binds a [2Fe-2S] cluster.
abstract: A linear cluster formulated as [S2MoS2CuS2MoS2](3-), a unique heterometallic cluster found in biological systems, was identified in a small monomeric protein (named as Orange Protein). The gene coding for this protein is part of an operon mainly present in strict anaerobic bacteria, which is composed (in its core) by genes coding for the Orange Protein and two ATPase proposed to contain Fe-S clusters. In Desulfovibrio desulfuricans G20, there is an ORF, Dde_3197 that encodes a small protein containing several cysteine residues in its primary sequence. The heterologously produced Dde_3197 aggregates mostly in inclusion bodies and was isolated by unfolding with a chaotropic agent and refolding by dialysis. The refolded protein contained sub-stoichiometric amounts of iron atoms/protein (0.5±0.2), but after reconstitution with iron and sulfide, high iron load contents were detected (1.8±0.1 or 3.4±0.2) using 2- and 4-fold iron excess. The visible absorption spectral features of the iron-sulfur clusters in refolded and reconstituted Dde_3197 are similar and resemble the ones of [2Fe-2S] cluster containing proteins. The refolded and reconstituted [2Fe-2S] Dde_3197 are EPR silent, but after reduction with dithionite, a rhombic signal is observed with gmax=2.00, gmed=1.95 and gmin=1.92, consistent with a one-electron reduction of a [2Fe-2S](2+) cluster into a [2Fe-2S](1+) state, with an electron spin of S=½. The data suggests that Dde_3197 can harbor one or two [2Fe-2S] clusters, one being stable and the other labile, with quite identical spectroscopic properties, but stable to oxygen.
authors.affiliation: UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Biplab K
authors.initials: BK
authors.lastname: Maiti
authors.affiliation: UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Isabel
authors.initials: I
authors.lastname: Moura
authors.affiliation: UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: José J G
authors.initials: JJG
authors.lastname: Moura
authors.affiliation: UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal. Electronic address: srp@fct.unl.pt.
authors.forename: Sofia R
authors.initials: SR
authors.lastname: Pauleta
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D045286:Desulfovibrio gigas / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D009876:Operon; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins
keywords: Desulfovibrio; Fe-S cluster reconstitution; Iron-sulfur cluster biosynthesis; Orange protein complex; Oxidative stress