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Updated: Dec 20, 2025

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons
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The human B12 trafficking protein CblC processes nitrocobalamin.

Romila Mascarenhas1, Zhu Li1, Carmen Gherasim2

  • 1Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan, USA.

The Journal of Biological Chemistry
|May 28, 2020
PubMed
Summary

Nitrosylcobalamin (NOCbl) oxidizes to nitrocobalamin (NO2Cbl), a substrate for the vitamin B12 trafficking enzyme CblC. CblC

Keywords:
MMACHCadenosylcobalamin (AdoCbl)enzyme kineticsmetalnitrosylcobalamin (NOCbl)processing chaperonethiolvitaminvitamin B12

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Quantification of Coenzyme A in Cells and Tissues
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Area of Science:

  • Biochemistry and Molecular Biology
  • Enzymology
  • Nutritional Biochemistry

Background:

  • Vitamin B12 (cobalamin) is essential, requiring complex intracellular trafficking to active cofactors.
  • CblC (MMACHC) is a key processing chaperone in vitamin B12 trafficking.
  • Mutations in CblC cause methylmalonic aciduria and homocystinuria.

Purpose of the Study:

  • To investigate the biochemical activities of CblC with nitrosylcobalamin (NOCbl) and its oxidation product, nitrocobalamin (NO2Cbl).
  • To elucidate the role of CblC in vitamin B12 metabolism and cellular processes.
  • To characterize the enzymatic mechanism of CblC's interaction with NO2Cbl.

Main Methods:

  • Biochemical assays to measure CblC's denitration and nitrite reductase activities.
  • Spectroscopic analysis of cobalamin derivatives.
  • Enzyme kinetics and characterization of wild-type and mutant CblC (R161G).

Main Results:

  • Nitrosylcobalamin (NOCbl) is unstable, rapidly oxidizing to nitrocobalamin (NO2Cbl) in air.
  • CblC catalyzes the GSH-dependent denitration of NO2Cbl to cob(II)alamin, which can be further oxidized or enter futile thiol cycling.
  • CblC exhibits nitrite reductase activity, converting cob(I)alamin and nitrite to NOCbl, and its denitration activity supports cell proliferation using NO2Cbl as a B12 source.

Conclusions:

  • CblC possesses novel nitrite reductase and denitration activities, expanding its known catalytic repertoire.
  • NO2Cbl is a substrate for CblC, and CblC's activity can utilize NO2Cbl as a source of vitamin B12.
  • The R161G mutation in CblC impairs its function, leading to stabilized cob(II)alamin and promoting futile cycling.