Purification and biophysical characterization of a new [2Fe-2S] ferredoxinfrom Azotobacter vinelandii, a putative [Fe-S] cluster assembly/repair protein

During the purification of site-directed mutant variants of Azotobacter vin elandii ferredoxin I (FdI), a pink protein, which was not observed in nativ e FdI preparations, appeared to associate specifically with variants that h ad mutations in ligands to FdI [Fe-S] clusters. That protein, which me desi gnate FdIV has now been purified. NH2-terminal sequence analysis revealed t hat the protein is the product of a previously described gene, herein desig nated fdxD, that is in the A. vinelandii iscSUA operon that encodes protein s involved in iron-sulfur cluster assembly or repair. An apoprotein molecul ar mass of 12,434.03 +/- 0.21 Da was determined by mass spectrometry consis tent with the known gene sequence, The monomeric protein was shown to conta in a single [2Fe-2S](2+/+) cluster by UV/visible, CD, and EPR spectroscopie s with a reduction potential of -344 mV versus the standard hydrogen electr ode. When overexpressed in Escherichia coli, recombinant FdIV holoprotein w as successfully assembled. However, the polypeptide of the recombinant prot ein was modified in some way such that the apoprotein molecular mass increa sed by 52 Da. Antibodies raised against FdIV and EPR spectroscopy were used to examine the relative levels of FdIV and FdI in various A. vinelandii st rains leading to the conclusion that FdIV levels appear to be specifically increased under conditions where another protein, NADPH:ferredoxin reductas e is also up-regulated. In that case, the fpr gene is known to be activated in response to oxidative stress. This suggests that the fdxD gene and othe r genes in the iron-sulfur cluster assembly or repair operon might be simil arly up-regulated in response to oxidative stress.