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.
Highlights
The periplasmic nitrate reduction (Nap)1 system has been found in many different bacteria, and several physiological
The subcellular localization of NapF is controversial due to the presence of a conserved twin arginine motif, which could be involved in the periplasmic targeting of the protein, this motif is not followed by a hydrophobic region [7]
Sequence comparison of the NapF proteins shows the presence of a conserved twin arginine motif at the Nterminal end, which could act as a possible signal for its translocation to the periplasm through the Tat pathway (Fig. 1A)
Summary
Bacterial strains R. sphaeroides DSM158 R. sphaeroides DSM158 (pFR86 Km1/2) E. coli JM109 E. coli DH5␣ E. coli XL1-blue. PSVB25 pGEM-T pKPD60 pQE32 pFR24 pALTER pALTER-PB pKPD60X pKPD60⌬ SX pKPD60B*X* pFR24 pQE32/napF. Host strain for plasmids carrying the pQE derivatives LacϪ, host strain for plasmids carrying the lacZ or phoA genes Host strain for plasmids carrying the phoA gene. AmpR, lacZ, f1ori, T7 promotera Cloning vector, AmpR, Lacϩ Poly-T cloning vector for PCR fragments, AmpR From pKK223–3 with a cycA81’-phoA fusion, AmpR Cloning vector carrying the His motif, AmpR 2.17-kb PstI-BamHI fragment with the napKEFD genes cloned into pUC18⌬ SalI, AmpR Site-directed mutagenesis vector, AmpR 2.17-kb PstI/BamHI fragment with the napKEFD genes cloned into pALTER, AmpR From pKPD60 with a napF26’-phoA fusion From pKPD60 with a deletion of the cycA sequence From pKPD60 with a napA41’-phoA fusion 2.17-kb PstI-BamHI fragment cloned into pUC18⌬ SalI, AmpR 0.8-kb BamHI fragment with the napFD genes cloned into pQE32 a AmpR, ampicillin-resistance marker
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