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 B ₁₂ and is found in Archaea as a short form (CbiX^S containing 120-145 amino acids) and in some bacteria as a longer version (CbiX^L containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiX^L 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 CbiX^L from both B. megaterium and Synechocystis. Sequence analysis of CbiX^L proteins reveals only two conserved cysteine residues within the CbiX^L 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.