Characterization of the cobaltochelatase CbiXL: evidence for a 4Fe-4S center housed within an MXCXXC motif.
CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 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.
Leech
Helen K
HK
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
Raux
Evelyne
E
McLean
Kirsty J
KJ
Munro
Andrew W
AW
Robinson
Nigel J
NJ
Borrelly
Gilles P M
GP
Malten
Marco
M
Jahn
Dieter
D
Rigby
Stephen E J
SE
Heathcote
Peter
P
Warren
Martin J
MJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016415:Sequence Alignment
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D008190:Lyases; C033211:cobaltochelatase