Ribonucleotide reductase is the only enzyme that catalyses de novo formation of deoxyribonucleotides and is thus a key enzyme in DNA synthesis. The radical-based reaction involves five cysteins. Two redox-active cysteines are located at adjacent antiparallel strands in a new type of ten-stranded alpha/beta-barrel, and two others at the carboxyl end in a flexible arm. The fifth cysteine, in a loop in the centre of the barrel, is positioned to initiate the radical reaction.

Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3'-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5´-diphosphates (NDPs) to 2´-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C439) in α2 by a stable diferric tyrosyl radical (Y122•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y122•→ W48→ Y356 in β to Y731→ Y730→ C439 in α. To study the details of this process, 3-aminotyrosine (NH2Y) has been site-specifically incorporated in place of Y356 of β. The resulting protein, Y356NH2Y-β2, and the previously-generated proteins Y731NH2Y-α2 and Y730NH2Y-α2 (NH2Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2–0.7 s−1. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH2Y-RNRs and not the result of co-purifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH2Y•s) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH2Y• formation is biphasic (kfast of 9–46 s−1 and kslow of 1.5–5.0 s−1) and kinetically-competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational-gating of NH2Y• formation, while the kcat of ~0.5 s−1 is proposed to be associated with rate-limiting oxidation by NH2Y• of the subsequent amino acid on the pathway during forward PCET. The Xray crystal structures of Y730NH2Y-α2 and Y731NH2Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y•) cofactor to initiate catalysis. The initiation process requires long range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y 122