Recombinant Escherichia coli biotin synthase is a [2Fe-2S](2+) protein in whole cells.
EPR and Mössbauer spectroscopies have been used to determine the type and properties of the iron-sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification. Difference EPR spectra of samples of whole cells from a strain over-expressing E. coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the [2Fe-2S](+) cluster of the E. coli iron-sulfur cluster assembly 2Fe ferredoxin, based on principal g-values, linewidths and relaxation behavior. Comparison of the Mössbauer spectra of whole cells with and without the bioB insertion revealed that the E. coli cells with over-expressed BioB contain an additional species that exhibits a spectrum identical to that of the [2Fe-2S](2+) cluster in purified recombinant BioB. The concentration of this [2Fe-2S](2+) species in the whole cell sample was quantified using a Mössbauer standard and found to be approximately 260 microM, which was comparable to the BioB protein concentration estimated for the cell paste. The results demonstrate that the [2Fe-2S](2+) cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over-expressed in an inactive form during aerobic growth.
Cosper
Michele Mader
MM
Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA.
Jameson
Guy N L
GN
Eidsness
Marly K
MK
Huynh
Boi Hanh
BH
Johnson
Michael K
MK
D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015204:Spectroscopy, Mossbauer; D013466:Sulfurtransferases / Q000737:chemistry / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D013466:Sulfurtransferases; C057985:biotin synthetase