The Cop-cyanocobamides obtained by cyanide extractions from several acetogenic bacteria were structurally characterized by ultraviolet/visible spectra, proton-nuclear-magnetic-resonance spectra and fast-atom-bombardment mass spectra. p-Cresolylcobamide was detected as a major corrinoid from Sporomusa ovata. This 'complete' corrinoid was isolated from an organism for the first time. Instead of the common Coa bases of the known and biologically active cobamides, p-cresolylcobamide contained a glycosidically bound cresolyl function that was unable to coordinate to the cobalt of the corrin ring. An additional, previously unknown corrinoid from natural sources, Coa-[a-(5-methoxy-6-methylbenzimidazolyl)]-Co~-cyanocobamide, was isolated along with vitamin B1 from Clostridium formicoaceticum. Both homoacetogenic eubacteria were grown on methanol and contained high amounts of corrinoids (> 950 nmol/g cell dry mass). Less corrinoid was isolated from Acetobacterium woodii and characterized as vitamin B12.

Methanobacterium thermoautotrophicum grown on mineral medium contains 120 nmol of Coa-(5-hydroxybenzimidazolyi)cobamides (derivaties of factor HI) per g of dry cell mass as the sole cobamide. The bacterium assimilated several corrinoids and bemzinidazole bases during autotrophic growth. The corrinoids were converted into factor Im; however, after three transfers in 5,6-dimethylbenzimidazole (200 uM)-supplemented mineral medium, derivatives of factor III were completely replaced by derivatives of vitamin B12, which is atypical for methanogens. The total cobamide content of these cells and their growth rate were not affected compared with factor I-containing cells. Therefore, the high cobamide content rather than a particular type of cobamide is required for metabolism of methanogens. Derivatives of factor m are not essential cofactors of cobamide-containing enzymes from methanogenic bacteria, but they are the result of a unique biosynthetic ability of these archaebacteria. The cobamide biosynthesis include unspecific enzymes, which made it possible either to convert non-species-derived comnoids into derivatives of factor m or to synthesize other types of cobamides than factor m. The cobamide biosynthesis is regulated by its end product. In addition, the uptake of extracellular cobamides is controlled, and the assimilated corrinoids regulate cellular cobamide biosynthesis. 124 Bq/nmol) in 0.5 ml of 2 M HCl. The solutions were incubated at 130°C for 2 h. After addition of 1 ml of concentration ammonia, the samples were twice flash evaporated to dryness and finally suspended in 500 ,ul of 80% chloroform-20% methanol. The 14C-labeled bases were then 3076 JOURNAL

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 < 2% (< 30 nmol/g dry cells) of the total corrinoids (> 2400 nmol/g dry cells) were in a low-spin Co(I1) 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(1) prior to this treatment. The cob(1)amide was quantified as Co(II1)-CH3 after methylation by iodomethane. More than 45% (1100 nmol/g dry cells) of the extractable corrinoids were in the methylated form, whereas nontreated cells revealed less then 1% (< 15 nmol/g dry cells) of light-sensitive corrinoids. EPR spectra of the Co(I1) complex exhibited a threefold N-hyperfine splitting in the g, region, which was similar to vitamin BIZ. Cells grown with [1,3-' 'Nz]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(1)amide is the major corrinoid complex in vivo and that it is stabilized by its protein(s). The Co(I1) ion of the prosthetic group was coordinated by one histidine residue of the apoprotein(s). The adenosyl-cobalamin in the holoenzyme is > 10" more active than the isolated prosthetic group [S]. Mechanistical aspects of the interaction, however, are still a matter of hypotheses [9 -121. Interactive corrinoid and protein structures are also considered to play a central role in human corrinoidbinding proteins like the intrinsic factors, transcobalamins and haptocorrins, as well as in corrinoid-transport proteins of bacterial membranes.Our recent corrinoid screening revealed eight different corrinoid derivatives which have been assigned to physiologically and evolutionary diverse bacterial strains [13]. Two of the corrinoids, p-cresolyl cobamide and phenolyl cobamide, lack an axial N-heterocyclic ligand, and hence they are 'base-off corrinoids. p-Cresolyl cobamide, but not phenolyl cobamide, was found to be essential for growth of Sporomusa ovata, and vitamin B12 could not substitute for p-cresolyl cobamide [14].With respect to its redox properties, the unique corrinoid presumably resembles the nucleotide-free aquo-cobinamide, rather than the 'base-on' corrinoids like vitamin B12.This paper reports on the redox states and the axial corrinoid ligand of p-cresolyl cobamide in the anaerobic bacterium S. ovata. Whole bacterial cells as harvested were analyzed by EPR spectroscopy, investigating the paramagnetic properties of the cob(I1)amides. The clear spectral difference between the 14N and the "N-labeled Co complexes was utilized to identify the cob(I1)amide ligand. The EPR-silent cob(1)amide was analyzed after alkylation, which yielded an alkyl-cob(II1)amide.

Phenolyl cobamide was isolated from cyanide extractions of the anaerobic eubactcrium Sporomusa civutu. The proposed corrinoid structure (Coa,Cob-(monocyano,monoaquo)-phenolyl cobamide) has been deduced from 'H NMR, fast-atom-bombardment mass spectroscopy and ultravioletivisible spectroscopy data. The complete corrinoid resembled p-cresolyl cobamide { Cox,Cop-(monocyano,monoaquo)-p-cresolyl cobamide), which recently has been obtained from cyanide extractions of the same bacterium. The structures and chemical properties of both cobamides with uncoordinated nucleotides differed significantly from those of vitamin BI2 {Cox-[a-(5,6-dimethylbenzimidazolyl)]-Co/3-cyanocobamide}. Spoiomu.su synthesized coenzymes of phenolyl co bamide and pcresolyl cobamide in considerable amounts of 400 nmol/g and 1700 nmol/g dry cells, respectively. More than 90% of the complete corrinoid pool of the homoacetogenic bacterium consisted of these two corrinoids, indicating that they are physiologically important coenzymes of the bacterial metabolism.Vitamin B,, is ubiquitous in both procaryotes and eucaryotes [I]. Several pure bacterial cultures synthesize vitamin B I 2 dcrivatives (see Table l), but each species appears to contain only one complete corrinoid , i.e. a cobamide with a heterocyclic base-containing Cox ligand. Some other cobamides with heterocyclic bases have been detected exclusively in cyanide extractions of sewage slugde, e.g. Coa-(clnaphthimidazolyl)]-Cop-cyanocobamide [2] and Cocr-(x-2-met1iylsulfinyladenine)-Cob-cyanocobamide [ 3 ] . Thcse corrinoids arc obtained also from cyanide extractions of propionibacterial fermentations, when the bacteria are grown with high amounts of the adequate heterocyclic basc functioning as a precursor of the Coct ligand. The method of manipulating the corrinoid metabolism in this way is reffered to as 'guided biosynthesis' of cobamides.Based on an imidazole function within these Cox ligands, a nucleotide 'trutw-effect' is considered to modulate corrinoidcoenzyme-catalyzed reactions. Thus, one investigated subject is the nucleotide-dependent change of the Cop-ligand Previously we discovered p-cresolyl cobamide in Sporom u m ovutu [lo]. The corrinoid lacks a cobalt-coordinated nucleotide base, but contains p-cresol r-0-glycosidically attached to the ribose. In solution, this aromatic function is unable to coordinate to the cobalt center of the corrin ring. Thus, the mechanism of the p-cresolyl cobamide function and the biosynthesis of the corrinoid attracted attention [ll].Phenylhydrogenobarnide has been proved in some photosynthetic bacteria [12]. The corrinoid is the cobalt-free form ('descobaltocorrinoid') of the phenolyl cobamide, described in this communication. The biological function and the biosynthesis of the descobaltocorrinoid are unknown, in part because the cobalt-containing purinyl cobamide and vitamin B1 [ 131, rather than phenolyl cobamide or p-cresolyl cobamide have been described in phototrophs. It is generally accepted that the cobalt centers are the essential catalyt...

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