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 widespread 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-dipyridyltreated NapA, we propose a role for NapF in assembling the [4Fe-4S] center of the catalytic subunit NapA.

Role of individual nap gene cluster products in NapC-independent nitrate respiration of Wolinella succinogenes

“…The NapF proteins from R. sphaeroides and E. coli have been described recently as cytoplasmic iron-sulfur proteins that are possibly involved in the incorporation of the NapA [4Fe-4S] cluster (Olmo-Mira et al, 2004;Nilavongse et al, 2006). In E. coli the napF gene was found to be dispensable for nitrate reduction using either glycerol or formate as the growth substrate (Potter & Cole, 1999).…”

“…While the napA and napD genes seem to be present throughout, NapF, NapL and NapM are encoded less frequently. NapF and NapD are predicted to be cytoplasmic proteins that function in NapA maturation (Berks et al, 1995;Olmo-Mira et al, 2004;Nilavongse et al, 2006). To date, NapL has been found only in nap operons of eproteobacteria, while NapM is a multihaem cytochrome c that is encoded in some nap operons lacking napB (Jepson et al, 2006).…”

Respiratory Nitrate Ammonification by Shewanella oneidensis MR-1

“…The identical residues include all four groups of four Cys residues involved in iron-sulfur cluster formation, as well as the characteristic IRPPW motif (38) near the amino terminus. The NapF protein is hypothesized to catalyze formation of the NapA iron-sulfur cluster (35). The possible involvement of the S. oneidensis MR-1 NapF protein in nitrate respiration remains to be determined.…”

“…The NapG and NapH proteins participate in electron transfer from ubiquinol (9), and the NapD and NapF proteins are involved in enzyme assembly (35,49). A variety of nap clusters with different gene contents and organizations are present in diverse bacterial species (28).…”

Structural diversity in twin-arginine signal peptide-binding proteins

“…19). The other proteins encoded by the nap operon have roles either in electron transfer to NapA (20) or in the biosynthesis of NapA (21,22). NapD has been shown to be essential for NapA-dependent periplasmic nitrate reductase activity in E. coli (23).…”