title: Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis.
abstract: A novel metalloprotein containing a unique [S2MoS2CuS2MoS2](3-) cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI-MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S-Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV-visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal. marta.carepo@dqoi.ufc.br.|Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Campus do Pici, CEP 60455-760, Fortaleza, CE, Brazil. marta.carepo@dqoi.ufc.br.
authors.forename: Marta S P
authors.initials: MS
authors.lastname: Carepo
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Cíntia
authors.initials: C
authors.lastname: Carreira
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Raquel
authors.initials: R
authors.lastname: Grazina
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Małgorzata E
authors.initials: ME
authors.lastname: Zakrzewska
authors.affiliation: Aix Marseille Université, CNRS, LCB UMR 7283, 13402, Marseille, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Dolla
authors.affiliation: Aix Marseille Université, CNRS, LCB UMR 7283, 13402, Marseille, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Aubert
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Sofia R
authors.initials: SR
authors.lastname: Pauleta
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: JJ
authors.lastname: Moura
authors.affiliation: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal. isabelmoura@fct.unl.pt.
authors.forename: Isabel
authors.initials: I
authors.lastname: Moura
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003300:Copper / Q000737:chemistry*; D003901:Desulfovibrio / Q000378:metabolism*; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D001426:Bacterial Proteins; D003300:Copper; D008982:Molybdenum
keywords: Desulfovibrio; Metal reconstitution; Mo–Cu cluster; Orange protein; Protein-assisted synthesis; Strict anaerobic bacteria; orp gene cluster