Chelatase enzymes selectively insert metals into modified tetrapyrroles along the biosynthetic pathways to the ‘pigments of life’ – cofactor F430, vitamin B12, heme, siroheme, etc. Chelatases are specific to their native metal and tetrapyrrole substrate, despite high structural similarity and evidence of a shared evolutionary ancestry between enzymes that select for different metals. The enzymes involved in and the timing of the critical metal insertion step of the aforementioned pigments are well-studied, but the origin of their metal specificities remains unknown. Furthermore, metal selectivity in vivo is dependent on a number of factors that are difficult to replicate in vitro, which has resulted in incorrectly assigned native metal substrates for some chelatases, such as the nickel chelatase CfbA, which was originally annotated CbiXS—a cobalt chelatase. CfbA is now thought to be the primordial class II chelatase, from which HemH, CbiK, and SirB are evolutionarily-derived. We have characterized the affinity of Archaeoglobus fulgidus CfbA for three naturally abundant metals that have shown activity with the chelatase: Fe(II), Co(II), and Ni(II). These experiments indicate nearly equal affinity for Co(II) and Ni(II), with an order of magnitude weaker affinity for Fe(II), suggesting that the nickel-specificity of CfbA has a different origin. In addition, the metal binding site of CfbA has been investigated using magnetic circular dichroism spectroscopy, which suggests that His10 and His74 bind an octahedral Ni(II).

Assessment of the CfbA metal binding site and its affinity for naturally-abundant metals

ACS Spring 2022

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