The acquisition of CoIIby the corrin component
of vitamin
B12follows one of two distinct pathways, referred to as
early or late CoIIinsertion. The late insertion pathway
exploits a CoIImetallochaperone (CobW) from the COG0523
family of G3E GTPases, while the early insertion pathway does not.
This provides an opportunity to contrast the thermodynamics of metalation
in a metallochaperone-requiring and a metallochaperone-independent
pathway. In the metallochaperone-independent route, sirohydrochlorin
(SHC) associates with the CbiK chelatase to form CoII-SHC.
CoII-buffered enzymatic assays indicate that SHC binding
enhances the thermodynamic gradient for CoIItransfer from
the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic
acida,c-diamide (HBAD) associates with the CobNST
chelatase to form CoII-HBAD. Here, CoII-buffered
enzymatic assays indicate that CoIItransfer from the cytosol
to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic
gradient for CoIIbinding. Notably, there is a favorable
gradient for CoIItransfer from the cytosol to the MgIIGTP-CobW metallochaperone, but further transfer of CoIIfrom the GTP-bound metallochaperone to the HBAD-CobNST chelatase
complex is thermodynamically unfavorable. However, after nucleotide
hydrolysis, CoIItransfer from the chaperone to the chelatase
complex is calculated to become favorable. These data reveal that
the CobW metallochaperone can overcome an unfavorable thermodynamic
gradient for CoIItransfer from the cytosol to the chelatase
by coupling this process to GTP hydrolysis.