Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B.

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