Characterization of the NifU and NifS Fe−S Cluster Formation Proteins Essential for Viability in Helicobacter pylori

Abstract

The Fe−S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O₂ atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains ∼1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S]^2+,+ cluster by characteristic UV−visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe−S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV−visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from l-cysteine, in agreement with its proposed role as a sulfur donor to Fe−S clusters. The results here indicate that the NifU type of Fe−S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe−S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe−S clusters.