Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B12

Tessa R Young; Evelyne Deery; Andrew W Foster; Maria Alessandra Martini; Deenah Osman; Martin J Warren; Nigel J Robinson

doi:10.1021/jacsau.3c00119

Two Distinct Thermodynamic Gradients for Cellular Metalation of Vitamin B₁₂

JACS Au. 2023 May 10;3(5):1472-1483. doi: 10.1021/jacsau.3c00119. eCollection 2023 May 22.

Authors

Tessa R Young^{1

2}, Evelyne Deery³, Andrew W Foster^{1

2}, Maria Alessandra Martini^{1

4}, Deenah Osman^{1

2}, Martin J Warren^{3

5}, Nigel J Robinson^{1

2}

Affiliations

¹ Department of Biosciences, Durham University, Durham DH1 3LE, U.K.
² Department of Chemistry, Durham University, Durham DH1 3LE, U.K.
³ School of Biosciences, University of Kent, Canterbury CT2 7NJ, U.K.
⁴ Department of Inorganic Spectroscopy, Max Planck Institute for Chemical Energy Conversion, 45470 Mülheim an der Ruhr, Germany.
⁵ Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, U.K.

Abstract

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.