Recombinant Escherichia coli biotin synthase is a [2Fe–2S]2+ protein in whole cells

FEBS Letters

Cosper, Michele Mader¹  Johnson, Michael K¹  Eidsness, Marly K¹  Jameson, Guy N.L²  Huynh, Boi Hanh² 

[1] Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA;Department of Physics, Emory University, Atlanta, GA 30322, USA

关键词: Biotin synthase;    BioB;    Iron–sulfur cluster;    Mössbauer whole cell;    EPR whole cell;    BioB;    gene product of bioB commonly referred to a biotin synthase;    SAM;    S-adenosyl-L-methionine;    Fd;    ferredoxin;   

DOI  :  10.1016/S0014-5793(02)03390-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 μM, 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.

【 授权许可】

Unknown