Preparation and characterization of cobalt-substituted anthrax lethal factor

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressing Bacillus megaterium cells in the presence of CoCl2, and (ii) direct exchange by treatment of zinc-LF with CoCl2. Independent of the method employed, the protein was found to contain one Co2+ per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co2+ ion to be five-coordinate, an observation similar to that reported for other Co2+-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co2+:TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF’s active site, which may be utilized in future studies to gain further insight into the enzyme’s mechanism and inhibitor interactions.

► Cobalt-substituted anthrax lethal factor (CoLF) is highly active.
► CoLF can be prepared by bio-assimilation and direct exchange.
► Lethal factor binds cobalt tightly.
► The electronic spectrum of CoLF reveals penta-coordination.
► Interaction of CoLF with thioglycolic acid follows a 2-step mechanism.