A divalent metal site in the small subunit of the manganese-dependent ribonucleotide 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 Brevibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I = 5/2,55Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence 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) spectrum indicates a protein-bound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.
Griepenburg
U
U
Institut für Mikrobiologie der Universität, Hannover, Germany.
Blasczyk
K
K
Kappl
R
R
Hüttermann
J
J
Auling
G
G
D001665:Binding Sites; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003352:Corynebacterium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D008345:Manganese / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*
D002413:Cations, Divalent; D008345:Manganese; D012264:Ribonucleotide Reductases