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

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.IMPORTANCEAnaerobic ammonium-oxidizing (anammox) bacteria contribute to the global nitrogen cycle by removing fixed nitrogen from the environment. They do so via the anaerobic oxidation of ammonium to dinitrogen gas, with nitrite as terminal electron acceptor and nitric oxide and hydrazine as free intermediates. This anammox energy metabolism is carried out by heme-containing enzymes, most of which are unique to anammox bacteria (i.e., hydrazine synthase and hydrazine dehydrogenase). The heme-containing enzymes rely on the availability of iron for their synthesis and functioning. Iron storage is thus expected to play an important role in anammox bacteria. Here we purify, identify, and characterize a bacterioferritin, an iron storage protein, from the anammox bacterium Kuenenia stuttgartiensis. We propose and discuss that bacterioferritin could play an important role in iron storage in K. stuttgartiensis and other anammox bacteria for potential use and reuse of iron in heme biosynthesis.