Purification, identification, and characterization of a bacterioferritin from the anammox bacterium Kuenenia stuttgartiensis

Anaerobic ammonium-oxidizing (anammox) bacteria convert ammonium and nitrite to nitrogen gas, with nitric oxide and hydrazine as intermediates. The anammox reaction takes place inside a dedicated cell compartment called the anammoxosome and is proposed to be coupled to energy conversion via a proton motive force over the anammoxosome membrane. This anammox energy metabolism is for a large part carried out by heme-containing enzymes, which depend on readily available iron for synthesis and proper functioning. Bacterioferritins are multimeric iron storage proteins. The genome of the model anammox species Kuenenia stuttgartiensis contains two putative bacterioferritin genes: KSMBR1_0066 and KSMBR1_0230. Here, we isolated and characterized K. stuttgartiensis bacterioferritin KSMBR1_0066 (KsBfr) and investigated the distribution of bacterioferritin homologs in anammox genomes. KsBfr was purified from K. stuttgartiensis cells and identified as KSMBR1_0066. A search for putative Bfr homologs in anammox Brocadiales genomes showed that most contain either one or two homologs and that these cluster in two distinct groups: bfr1 and bfr2. KSMBR1_066 clustered with bfr2. Purified KsBfr had an 18-kDa monomer size, consisted of 14-nm-wide particles with a 6-nm electron-dense core, and contained heme b with absorption maxima at 423, 527, and 557 nm when reduced. As-isolated KsBfr could be unloaded and subsequently reloaded with iron. We propose that KsBfr is involved in iron storage in K. stuttgartiensis cells and speculate that anammox bacterioferritins could play a role in iron storage for use and reuse of iron in heme biosynthesis.