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

Abstract

Archaeoglobus fulgidus CbiXS is a small (16 kD) 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 CbiXS 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 metal-free and metal-bound forms of CbiXS with electrospray ionization spectrometry (ESI-MS) indicated that CbiXS binds a divalent metal substrate using two protonatable amino acids. Additional characterization of CbiXS using UV circular dichroism spectroscopy suggest that the protein remains folded in the pH 6-8 range. Using established competitive binding assays, it was discovered that divalent metal binding by CbiXS 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 CbiXS as a novel cobalt chelatase, distinct from CbiK or CbiXL. Based upon the ESI-MS and pH-dependent metal affinity data, in addition to available structural data, Co(II) is most likely ligated by His10 and His74.