Enzymes with a dimetalcarboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O-2) activation. Recently described proteins bind a manganeseiron cofactor (MnFe) instead of the classical diiron cofactor (FeFe). Determination of atomic-level differences of homo- versus hetero-bimetallic cofactors is crucial to understand their diverse redox reactions. We studied a ligand-binding oxidase from the bacterium Geobacillus kaustophilus (R2lox) loaded with a FeFe or MnFe cofactor, which catalyzes O-2 reduction and an unusual tyrosinevaline ether cross-link formation, as revealed by X-ray crystallography. Advanced X-ray absorption, emission, and vibrational spectroscopy methods and quantum chemical and molecular mechanics calculations provided relative Mn/Fe contents, X-ray photoreduction kinetics, metalligand bond lengths, metalmetal distances, metal oxidation states, spin configurations, valence-level degeneracy, molecular orbital composition, nuclear quadrupole splitting energies, and vibrational normal modes for both cofactors. A protonation state with an axial water (H2O) ligand at Mn or Fe in binding site 1 and a metal-bridging hydroxo group (OH) in a hydrogen-bonded network is assigned. Our comprehensive picture of the molecular, electronic, and dynamic properties of the cofactors highlights reorientation of the unique axis along the MnOH2 bond for the Mn1(III) JahnTeller ion but along the Fe-mu OH bond for the octahedral Fe1(III). This likely corresponds to a more positive redox potential of the Mn(III)Fe(III) cofactor and higher proton affinity of its mu OH group. Refined model structures for the Mn(III)Fe(III) and Fe(III)Fe(III) cofactors are presented. Implications of our findings for the site-specific metalation of R2lox and performance of the O-2 reduction and cross-link formation reactions are discussed.