Proteins in the Ferritin-like superfamily are characterized by a four alpha-helical structural motif. These proteins are distributed across all three kingdoms of life and perform a wide range of functions. Several members in this protein superfamily can activate dioxygen using a di-metal active site coordinated by four carboxylate and two histidine amino acid residues. The resulting diverse set of dioxygen activated intermediates is used in nature to perform complex redox chemical reaction in cells. The R2 subunit of class I Ribonucleotide reductase and soluble Methane monooxygenase are the most well-characterized groups of proteins in this superfamily. Upon oxygen (or reduced-oxygen) activation of the di-metal site, the R2 subunit can generate a catalytic radical required for the conversion of ribonucleotides to deoxyribonucleotides, while soluble Methane monooxygenase can oxidize methane to methanol in an alternative form of carbon assimilation.

The work presented in this thesis aims to better understand metal selectivity, working and the regulation of substrate specificity in various proteins of the Ferritin-like superfamily, and the development of a novel method to study radiation-sensitive intermediates. The papers discussed in this thesis present crystallographic and spectroscopic studies of several Ferritin-like superfamily proteins.

In paper I, the assembly mechanisms of the heterodinuclear manganese-iron cofactor in a class Ic R2 protein and an R2-like ligand-binding oxidase are compared. Paper II presents the discovery of a novel radical-generating subunit subclass of Ribonucleotide reductase in Mollicutes, including mycoplasma pathogens, that breaks the paradigm of metal requirement for radical translocation and catalysis. This new subclass, denoted class Ie, is shown to instead use an unprecedented modified tyrosine DOPA residue in its four-helix bundle for radical translocation and storage. Paper III presents a new X-ray free-electron laser sample delivery system that combines acoustic droplet ejection with a drop-on-tape setup, allowing simultaneous multimodal X-ray diffraction and X-ray emission data collection. This setup is also shown to support photochemical and chemical activation of catalysis in crystals, allowing the study of radiation-sensitive transient reaction intermediates. We used this setup in paper IV to solve the first radiation damage-free crystallographic structures of the soluble methane monooxygenase hydroxylase and its regulatory subunit complex from Methylosinus trichosporium OB3b. The high-resolution crystal structures of the complex, in both di-ferrous and di-ferric oxidation states, illustrate the structural reorganization in the hydroxylase subunit upon binding to the regulatory subunit.

These results illustrate the functional range and flexibility in the Ferritin-like protein superfamily. Including the distinctive metal discrimination in heterodinuclear metalloproteins, influencing substrate specificity in sMMO, and using a novel metal-free DOPA radical to catalyze ribonucleotide reduction in the class Ie R2 subclass. Experiments using the novel ADE-DOT setup also showed promising progress towards determining the highly sought-after structures of di-metal oxygen activated intermediates such as X and Q in subclass Ia R2 and sMMO, respectively.