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Applications of the 57Co EMS technique in biology-related fields have so far been rare, primarily owing to the necessity of using 57Co-containing radioactive compounds in samples under study. However, cobalt as a trace biogenic element is involved as a cofactor in a number of enzymes. In our earlier work, for the first time we successfully realised the idea of substituting 57Co2+ (as an EMS-active probe) for native cations in cation-binding sites (CBS) within the enzyme active centres, using glutamine synthetase (GS) as a model metalloenzyme. The resulting emission spectra showed separate contributions from the two structurally different CBS present in each GS active centre (there are 12 identical active centres per dodecameric GS molecule). We further applied 57Co EMS to probing 57Co2+-doped GS covalently modified by adenylylation, as well as in the presence of Mn2+ as an additional EMS-“invisible” cofactor. The emission spectra showed Mn2+-induced 57Co2+ redistribution between the two CBS and changes in the 57Co2+ coordination related to GS activity regulation.