title: NapF is a cytoplasmic iron-sulfur protein required for Fe-S cluster assembly in the periplasmic nitrate reductase.
abstract: The periplasmic nitrate reductase (Nap) is wide-spread in proteobacteria. NapA, the nitrate reductase catalytic subunit, contains a Mo-bisMGD cofactor and one [4Fe-4S] cluster. The nap gene clusters in many bacteria, including Rhodobacter sphaeroides DSM158, contain an napF gene, disruption of which drastically decreases both in vitro and in vivo nitrate reductase activities. In spite its importance in the Nap system, NapF has never been characterized biochemically, and its role remains unknown. The NapF protein has four polycysteine clusters that suggest that it is an iron-sulfur-containing protein. In the present study, a His(6)-tagged NapF protein was overproduced in Escherichia coli and purified anaerobically. The purified NapF protein was used to obtain polyclonal antibodies raised in rabbit, and cellular fractionation of R. sphaeroides followed by immunoprobing with anti-NapF antibodies revealed that the native NapF protein is located in the cytoplasm. This contrasts with the periplasmic location of the mature NapA. However, NapA could not be detected in an isogenic napF(-) strain of R. sphaeroides. The His(6)-tagged NapF protein displayed spectral properties indicative of Fe-S clusters, but these features were rapidly lost, suggesting cluster lability. However, reconstitution of the Fe-S centers into the apo-NapF protein was achieved in the presence of Azotobacter vinelandii cysteine desulfurase (NifS), and this allowed the recovery of nitrate reductase activity in NapA protein that had previously been treated with 2,2'-dipyridyl to remove the [4Fe-4S] cluster. This activity was not recovered in the absence of NapF. Taking into account the cytoplasmic localization of NapF, the presence of labile Fe-S clusters in the protein, the napF(-) strain phenotype, and the NapF-dependent reactivation of the 2,2'-dipyridyl-treated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Campus de Rabanales, Edificio Severo Ochoa, 1 Planta, Universidad de Córdoba, Córdoba 14071, Spain.
authors.forename: M Francisca
authors.initials: MF
authors.lastname: Olmo-Mira
authors.forename: Mónica
authors.initials: M
authors.lastname: Gavira
authors.forename: David J
authors.initials: DJ
authors.lastname: Richardson
authors.forename: Francisco
authors.initials: F
authors.lastname: Castillo
authors.forename: Conrado
authors.initials: C
authors.lastname: Moreno-Vivián
authors.forename: M Dolores
authors.initials: MD
authors.lastname: Roldán
mesh_terms: D000595:Amino Acid Sequence; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000201:enzymology / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013053:Spectrophotometry; D013455:Sulfur / Q000378:metabolism*; D013997:Time Factors
chemical_list: D033903:Periplasmic Proteins; D013455:Sulfur; D007501:Iron; D009565:Nitrate Reductases; D050901:Nitrate Reductase