Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B₁₂

The acquisition of Co^II by the corrin component of vitamin B₁₂ follows one of two distinct pathways, referred to as early or late Co^II insertion. The late insertion pathway exploits a Co^II metallochaperone (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 Co^II-SHC. Co^II-buffered enzymatic assays indicate that SHC binding enhances the thermodynamic gradient for Co^II transfer from the cytosol to CbiK. In the metallochaperone-dependent pathway, hydrogenobyrinic acid a,c-diamide (HBAD) associates with the CobNST chelatase to form Co^II-HBAD. Here, Co^II-buffered enzymatic assays indicate that Co^II transfer from the cytosol to HBAD-CobNST must somehow traverse a highly unfavorable thermodynamic gradient for Co^II binding. Notably, there is a favorable gradient for Co^II transfer from the cytosol to the Mg^IIGTP-CobW metallochaperone, but further transfer of Co^II from the GTP-bound metallochaperone to the HBAD-CobNST chelatase complex is thermodynamically unfavorable. However, after nucleotide hydrolysis, Co^II transfer 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 Co^II transfer from the cytosol to the chelatase by coupling this process to GTP hydrolysis.