Evidence for a super-reduced cobamide as the major corrinoid fraction in vivo and a histidine residue as a cobalt ligand of the p-cresolyl cobamide in the acetogenic bacterium Sporomusa ovata.

The redox state of cobalt in p-cresolyl cobamide and one of its axial ligands were determined by EPR spectroscopy of Sporomusa ovata as harvested. The analyses revealed that less than 2% (less than 30 nmol/g dry cells) of the total corrinoids (greater than 2400 nmol/g dry cells) were in a low-spin Co(II) complex. The amount increased to about 15% (190-450 nmol/g dry cells) upon partial oxidation by air, indicating that the original valence state of cobalt was a Co(I) prior to this treatment. The cob(I)amide was quantified as Co(III)-CH3 after methylation by iodomethane. More than 45% (1100 nmol/g dry cells) of the extractable corrinoids were in the methylated form, whereas non-treated cells revealed less than 1% (less than 15 nmol g dry cells) of light-sensitive corrinoids. EPR spectra of the Co(II) complex exhibited a threefold N-hyperfine splitting in the gz region, which was similar to vitamin B12. Cells grown with [1.3-15N2]histidine showed a twofold N-hyperfine splitting, demonstrating that the axial N ligand of the corrinoid was derived from the imidazole group of histidine. It is concluded that the super-nucleophilic p-cresolyl cob(I)amide is the major corrinoid complex in vivo and that it is stabilized by its protein(s). The Co(II) ion of the prosthetic group was coordinated by one histidine residue of the apoprotein(s).