A DIVALENT METAL SITE IN THE SMALL-SUBUNIT OF THE MANGANESE-DEPENDENTRIBONUCLEOTIDE REDUCTASE OF CORYNEBACTERIUM AMMONIAGENES

Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly Brevib acterium) ammoniagenes ATCC 6872 is no longer considered as a simple a nalogue of the aerobic Fe-RRase of Escherichia coli but as the prototy pe of the class IV enzymes (I). Deliberate dissociation of the Mn-RRas e holoenzyme and an improved sample preparation of the dimeric CA2 pro tein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K , a distinct hyperfine sextet (I = 5/2, Mn-55) pattern with a weak zer o-field splitting was detected in the CA2 protein prepared from mangan ese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from m anganese-depleted cells devoid of this enzymatic activity. The presenc e of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) sp ectrum indicates a protein-bound manganese which was modified in the p resence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDT A. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.