A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774, grown with nitrate as the terminal electron acceptor, which is the first example of a bacterioferritin from a strict anaerobic organism. This new bacterioferritin was isolated mainly as a 24-mer of 20 kDa identical subunits, containing 0.5 noncovalently bound heme and 2 iron atoms per monomer. Although its N-terminal sequence is significantly homologous with ferritins from other microorganisms and the ligands to the di-iron ferroxidase center are conserved, it is one of the most divergent bacterioferritins so far characterized. Also, in contrast to all other known bacterioferritins, its heme is not of the B type; its chromatographic behavior is identical to that of iron uroporphyrin. Thus, D. desulfuricans bacterioferritin appears to be the second example of a protein unexpectedly containing this heme cofactor, or a closely related porphyrin, after its finding in Desulfovibrio gigas rubredoxin:oxygen oxidoreductase ¿Timkovich, R., Burkhalter, R. S., Xavier, A. V., Chen, L., and Le Gall, J. (1994) Bioorg. Chem. 22, 284-293. The oxidized form of the protein has a visible spectrum characteristic of low-spin ferric hemes, exhibiting a weak absorption band at 715 nm, indicative of bis-methionine heme axial coordination; upon reduction, the alpha-band appears at 550 nm and a splitting of the Soret band occurs, with two maxima at 410 and 425 nm. The heme center has a reduction potential of 140 +/- 10 mV (pH 7.6), a value unusually high compared to that of other bacterioferritins (ca. -200 mV).