Metal tetrapyrroles play an essential role in several biological systems such as photosynthesis and methanogensis. As metal tetrapyrroles are not readily available in nature, organisms have developed complex biosynthetic pathways for these earth-abundant metal catalysts. The critical step of metal tetrapyrrole biosynthesis is metal insertion that is catalyzed by a chelatase enzyme specific for each metal-tetrapyrrole combination. However, the substrate scope of metal-tetrapyrrole combinations is limited to naturally-occurring products. Here, we have shown that the archaeal chelatase CfbA is able to catalyze the formation of the catalyst cobalt protoporphyrin IX under mild, aqueous conditions in vitro. This represents a significant expansion of the substrate scope for CfbA, which catalyzes nickel insertion into sirohydrochlorin in vivo. Low-temperature magnetic circular dichroism characterization of cobalt-bound CfbA indicates that the enzyme can bind a high-spin cobalt with an octahedral coordination geometry. UV/Vis absorption spectroscopy was used to assess tetrapyrrole binding by CfbA, as well as the catalytic activity of CfbA for divalent metal insertion into the tetrapyrrole substrates. Several tetrapyrroles exhibit band shifts that indicate CfbA binding alters the electronic structure of the substrate. Activity assays demonstrate that CfbA catalyzes the insertion of divalent metals into several tetrapyrroles at a rate faster than the enzyme-free reaction. CfbA has been shown to insert multiple 3d metals into distinct tetrapyrroles implying that CfbA is a promising starting point for the development of artificial chelatases.

Assessing the substrate scope of the chelatase CfbA

2019 Spring National Meeting

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