For deoxyribonucleotide synthesis during anaerobic growth, Escherichia coli cells depend on an oxygen-sensitive class III ribonucleotide reductase. The enzyme system consists of two proteins: protein ␣, on which ribonucleotides bind and are reduced, and protein ␤, of which the function is to introduce a catalytically essential glycyl radical on protein ␣. Protein ␤ can assemble one [4Fe-4S] center per polypeptide enjoying both the [4Fe-4S] 2؉ and [4Fe-4S] 1؉ redox state, as shown by iron and

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... fide analysis, Mö ssbauer spectroscopy (␦ ‫؍‬ 0.43 mm⅐s ؊1 , ⌬E Q ‫؍‬ 1.0 mm⅐s ؊1 , [4Fe-4S] 2؉ ), and EPR spectroscopy (g ‫؍‬ 2.03 and 1.93, [4Fe-4S] 1؉ ). This iron center is sensitive to oxygen and can decompose into stable [2Fe-2S] 2؉ centers during exposure to air. This degraded form is nevertheless active, albeit to a lesser extent because of the conversion of the cluster into [4Fe-4S] forms during the strongly reductive conditions of the assay. Furthermore, protein ␤ has the potential to activate several molecules of protein ␣, suggesting that protein ␤ is an activating enzyme rather than a component of an ␣ 2 ␤ 2 complex as previously claimed. Ribonucleotide reductases (RNRs) 1 catalyze the reduction of ribonucleotides into deoxyribonucleotides and thus provide the cell with a balanced supply of the DNA precursors (1-3). Escherichia coli uses different ribonucleotide reductases during aerobic and anaerobic growth. The active form of the anaerobic enzyme (class III RNR) is characterized by the presence of a catalytically essential glycyl radical and an iron-sulfur center as well as the requirement for formate as the hydrogen donor (4 -6). It is found in other anaerobically growing microorganisms among bacteria, phages, and methanogens (7) . The anaerobic RNR was originally isolated as a dimeric ␣ 2 (160 kDa) inactive form that could be activated by anaerobic incubation with a complex activating system consisting of Sadenosylmethionine (AdoMet), a reducing system (NADPH, flavodoxin reductase, and flavodoxin), and an additional 17-kDa ␤ protein, provisionally called activase (8, 9) . During the reaction, a radical is introduced at a specific glycine residue (Gly-681) of protein ␣. The activated protein ␣, encoded by the nrdD gene, thus contains the glycyl radical, the substrate site, and two additional sites where allosteric effectors (deoxyribo-nucleotides) bind and regulate the activity (4, 10 -12). The recently determined three-dimensional structure of a mutant form of the enzyme from bacteriophage T4, in which the essential glycine has been changed to an alanine, suggests that the function of the radical is to abstract a hydrogen atom from an adjacent cysteine close to the substrate (13). The resulting thiyl radical is then supposed to initiate the reaction by removing the 3Ј-hydrogen atom of the ribose (14). How reduction by formate and formation of the deoxyribonucleotide proceed from the sugar radical remains to be established. The small ␤ protein, encoded by the nrdG gene, proved to be an unusual enzyme. First, in solution in the absence of the large protein, it occurred in a monomer-polymer equilibrium, with ␤ and ␤ 2 being the major species. The addition of protein ␣ 2 shifts the equilibrium to the ␤ 2 form and results in a very tight 1:1 complex between dimers of the two proteins, as shown from sucrose gradient centrifugation (5) and from the impossibility of separating them during gel filtration or by affinity chromatography on dATP-Sepharose gel, on which only protein ␣ can bind because of its affinity for dATP (5, 10). It was thus concluded that ␤ 2 was not an activating enzyme but rather a component of the system and that the anaerobic ribonucleotide reductase had an ␣ 2 ␤ 2 structure (5). Second, whereas the presence of an iron-sulfur center was suggested early from the light absorption properties of the enzyme and from iron and sulfide analysis (15), very little iron could be retained during purification of the protein. However, treatment of the ␤ protein with ferrous iron and sulfide generated a well defined [2Fe-2S] 2ϩ cluster, as shown from Mössbauer and Raman resonance spectroscopy (16). Third, EPR and Mössbauer spectroscopies of the protein after reduction with photoreduced deazaflavin or dithionite showed that the reduced centers were almost exclusively [4Fe-4S] cubane clusters (5, 16). The reductive [2Fe-2S] to [4Fe-4S] conversion is a remarkable reaction, even though it has been recently also observed with other iron-sulfur proteins, such as the transcription factor FNR, the activating enzyme of the pyruvate formate-lyase and biotin synthase (17) (18) (19) . Whether the [4Fe-4S] center, in the reduced anaerobic ribonucleotide reductase, was at the interface of two ␤ polypeptide chains was first suggested as a likely possibility but not firmly established experimentally (5). Fourth, formation of the glycyl radical was shown to depend on the one-electron reduction of S-adenosylmethionine by the reduced [4Fe-4S] 1ϩ center (20). It was proposed that reduced AdoMet can undergo homolysis of the S-C(5Ј-deoxyadenosyl) bond to generate methionine and the 5Ј-deoxyadenosyl radical, presumably responsible for abstraction of the hydrogen atom of the glycine residue. Here we report evidence that previous models for the iron center of RNR need to be revised. As a matter of fact, we show,