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Characterization of the cobaltochelatase CbiXL: Evidence for a 4Fe-4S center housed within an MXCXXC motif

Lookup NU author(s): Professor Nigel Robinson, Dr Gilles Borrelly

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Abstract

CbiX is a cobaltochelatase required for the biosynthesis of vitamin B 12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution. The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center. The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90. The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center. Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B. megaterium and Synechocystis. Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif. Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement. The potential physiological role of the iron-sulfur center is discussed.


Publication metadata

Author(s): Leech HK, Raux E, McLean KJ, Munro AW, Robinson NJ, Borrelly GPM, Malten M, Jahn D, Rigby SEJ, Heathcote P, Warren MJ

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2003

Volume: 278

Issue: 43

Pages: 41900-41907

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology

URL: http://dx.doi.org/10.1074/jbc.M306112200

DOI: 10.1074/jbc.M306112200

PubMed id: 12917443


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