Listeria monocytogenes (Lmo) is a Gram-positive intracellular pathogen that causes life-threatening infections in humans. This bacterium utilizes a class I ribonucleotide reductase (RNR) for aerobic growth to catalyze the reduction of nucleotides to deoxynucleotides, which is essential for DNA synthesis and cell proliferation. The Lmo RNR was previously proposed to utilize a diferric-tyrosyl radical (Y•) cofactor, like canonical “class Ia” RNRs, to initiate substrate reduction via a proton-coupled electron transfer reaction. However, the Lmo RNR gene cluster also contains a gene encoding a putative NrdI protein, a flavodoxin-like protein that, in class Ib and Ie RNR subclasses, is proposed to react with O2 to generate superoxide for cofactor assembly; this suggests that Lmo RNR may employ a different cofactor than originally proposed.
Here, we demonstrate that the Lmo RNR can form a diferric-Y• cluster, which assembles rapidly but exhibits low enzymatic activity. Further investigation also revealed the protein’s ability to form a manganese-based cofactor with nearly quadruple the activity of the iron cofactor, although Y• formation proceeds more slowly (t1/2 = 10.6 minutes). The dimanganese-tyrosyl radical (Y•) demonstrates unusually fast relaxation behavior, as revealed through EPR spectroscopy, setting it apart from previously studied class Ib cofactors. The ability of Lmo class I RNR to utilize both iron and manganese cofactors may suggest its adaptability to operate effectively under metal-limiting conditions during different stages of the infection cycle.