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ABSTRACT The biomineralization pathway of magnetite in magnetotactic bacteria is still poorly understood and a matter of intense debates. In particular, the existence, nature, and location of possible mineral precursors of magnetite are not clear. One possible precursor has been suggested to be ferritin-bound ferrihydrite. To clarify its role for magnetite biomineralization, we analyzed and characterized ferritin-like proteins from the magnetotactic alphaproteobacterium Magnetospirillum gryphiswaldense MSR-1, employing genetic, biochemical, and spectroscopic techniques. Transmission Mössbauer spectroscopy of the wild type (WT) and a bacterioferritin (bfr) deletion strain uncovered that the presence of ferrihydrite in cells is coupled to the presence of Bfr. However, bfr and dps deletion mutants, encoding another ferritin-like protein, or even mutants with their codeletion had no impact on magnetite formation in MSR-1. Thus, ferritin-like proteins are not involved in magnetite biomineralization and Bfr-bound ferrihydrite is not a precursor of magnetite biosynthesis. Using transmission electron microscopy and bacterial two-hybrid and electrophoretic methods, we also show that MSR-1 Bfr is an atypical representative of the Bfr subfamily, as it forms tetraeicosameric complexes from two distinct subunits. Furthermore, our analyses revealed that these subunits are functionally divergent, with Bfr1 harboring a ferroxidase activity while only Bfr2 contributes to heme binding. Because of this functional differentiation and the poor formation of homooligomeric Bfr1 complexes, only heterooligomeric Bfr protects cells from oxidative stress in vivo. In summary, our results not only provide novel insights into the biomineralization of magnetite but also reveal the unique properties of so-far-uncharacterized heterooligomeric bacterioferritins. IMPORTANCE Magnetotactic bacteria like Magnetospirillum gryphiswaldense are able to orient along magnetic field lines due to the intracellular formation of magnetite nanoparticles. Biomineralization of magnetite has been suggested to require a yet-unknown ferritin-like ferrihydrite component. Here, we report the identification of a bacterioferritin as the source of ferrihydrite in M. gryphiswaldense and show that, contrary to previous reports, bacterioferritin is not involved in magnetite biomineralization but required for oxidative stress resistance. Additionally, we show that bacterioferritin of M. gryphiswaldense is an unusual member of the bacterioferritin subfamily as it is composed of two functionally distinct subunits. Thus, our findings extend our understanding of the bacterioferritin subfamily and also solve a longstanding question about the magnetite biomineralization pathway.