Purification and biophysical characterization of a new [2Fe-2S] ferredoxin from Azotobacter vinelandii, a putative [Fe-S] cluster assembly/repair protein.
During the purification of site-directed mutant variants of Azotobacter vinelandii ferredoxin I (FdI), a pink protein, which was not observed in native FdI preparations, appeared to associate specifically with variants that had mutations in ligands to FdI [Fe-S] clusters. That protein, which we designate FdIV, has now been purified. NH(2)-terminal sequence analysis revealed that the protein is the product of a previously described gene, herein designated fdxD, that is in the A. vinelandii iscSUA operon that encodes proteins involved in iron-sulfur cluster assembly or repair. An apoprotein molecular mass of 12,434.03 +/- 0.21 Da was determined by mass spectrometry consistent with the known gene sequence. The monomeric protein was shown to contain a single [2Fe-2S](2+/+) cluster by UV/visible, CD, and EPR spectroscopies with a reduction potential of -344 mV versus the standard hydrogen electrode. When overexpressed in Escherichia coli, recombinant FdIV holoprotein was successfully assembled. However, the polypeptide of the recombinant protein was modified in some way such that the apoprotein molecular mass increased 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 strains leading to the conclusion that FdIV levels appear to be specifically increased under conditions where another protein, NADPH:ferredoxin reductase 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 other genes in the iron-sulfur cluster assembly or repair operon might be similarly up-regulated in response to oxidative stress.
Jung
Y S
YS
Department of Molecular Biology, University of California, Irvine, California 92697, USA.
Gao-Sheridan
H S
HS
Christiansen
J
J
Dean
D R
DR
Burgess
B K
BK
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000502:physiology; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D004269:DNA, Bacterial; D005288:Ferredoxins