An Active Dimanganese(III)-Tyrosyl Radical Cofactor in Escherichia coli Class Ib Ribonucleotide Reductase

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: alpha 2 (NrdE), where nucleotide reduction occurs, and beta 2 (NrdF), which contains an unidentified metal loco factor that initiates nucleotide reduction. nrdE and nrdF are found In an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (HIS)(6)-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with Mn-II and incubated with fully reduced NrdI (NrdI(hq)) and O-2. Active RNR was rapidly produced with 0.25 +/- 0.03 tyrosyl radical (Y.) per beta 2 and a specific activity of 600 Units/mg. EPR and blochemical studies or the reconstituted cofactor suggest it is Mn-2(III)-Y., which we propose is generated by Mn-2(II)-NrdF reacting with two equivalents of HO2-, produced by reduction of O-2 by NrdF-bound Ni-dI(hq). In the absence of NrdI(hq), with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with Fe-II and Incubated with O-2, in the presence or absence of NrdI(hq) gave 0.2 and 0.7 Y./beta 2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdI(hq) hinders Fe-2(III)-Y. cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the Mn-2(III)-Y. cofactor, not the diferric-Y. one, is the active metallocofactor in vivo.