Background: Ferritin stores iron by ferroxidation to form a mineral core, enabling diatom blooms upon iron input.Results: Ferrous iron binds solely to ferroxidase site A anaerobically. Ferroxidation kinetics has two observed phases.Conclusion: Ferrous iron and dioxygen binding to the di-iron ferroxidase site is stepwise.Significance: Iron storage by ferritins requires a coordinated binding of iron and dioxygen.

Background: Iron storage by ferritin enables diatom bloom upon iron input.Results: Ferroxidase center variants show faster iron mineralization and rate of post-oxidation reorganization of iron.Conclusion: Glu-130 and Glu-44 regulate the flux of iron through the ferroxidase center.Significance: Optimization of ferritin for iron oxidation but not mineralization suggests an iron buffering function in addition to long-term iron storage.

Metals are essential for all microorganisms as they are required as cofactors of enzymes that mediate metabolic processes that are indispensable for cellular energy production and growth. Some metals, such as zinc, are readily bound and serve as key structural elements of many macromolecules. Thus, to grow, microorganisms have an essential quota for several metals. The catalytic and other chemical properties of metals that microorganisms value create issues for metal management. Due to their high affi nity for amino acids and their reactive nature, uptake, intracellular transport, and storage of metals are mediated by tightly regulated proteins. Protein chaperones function to supply some specifi c metals to sites of utilization and, in some cases, storage. In particular, iron is diffi cult to acquire and is stored as a mineral in protein nanocages. Other metals, when present in excess, induce the expression of export systems to maintain a defi ned intracellular concentration of readily exchangeable metal.

The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.

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