Archaeoglobus fulgidus CbiX^S is a Cobalt Chelatase Where Co(II) is Ligated by His10 and His74

Archaeoglobus fulgidus CbiX^S is a small (16 kDa) class II chelatase with structural homology to the CfbA nickel chelatase required for the biosynthesis of cofactor F₄₃₀. However, the biochemical function of A. fulgidus CbiX^S has remained a point of confusion in the literature, with assignments as either a cobalt or a nickel chelatase. In this work, we analyze the genome of A. fulgidus revealing that the organism lacks the cofactor F₄₃₀ biosynthetic pathway but contains a nearly complete vitamin B₁₂ biosynthetic pathway, lacking a cobalt chelatase. Characterization of CbiX^S using UV circular dichroism spectroscopy suggests that the protein remains folded in the pH 6-8 range. Using established competitive binding assays, it was discovered that divalent metal binding by CbiX^S is a notable exception to the Irving-Williams series, with Co(II) and Ni(II) binding affinities within error of one another at pH 7.0. Additional competitive binding experiments at pH 6.0 and 8.0 suggest that Co(II) and Ni(II) are ligated by amino acids with side-chain pKa values between 6.0 and 7.0. Collectively, these data lead to the assignment of CbiX^S as a novel cobalt chelatase, distinct from CbiK or CbiX^L. Based upon site-directed mutagenesis, Co(II) is most likely ligated by His10 and His74.