# Structural Biochemistry/Nucleic Acid/Nitrogenous Bases/Ribonucleotide Reductase

Ribonucleotide reductase (or RNR) is the enzyme responsible for catalyzing the reduction of ribonucleotides to deoxyribonucleotides. These deoxyribonucleotides can then be utilized by the cell in DNA replication. Additionally, because of the role RNR plays in the formation of deoxyribonucleotides, RNRs are responsible for regulating the rate of DNA synthesis within the cell.

## Classes of RNR

* Class I: Class I RNRs consist two subgroups (Ia, Ib, and Ic) which differ only slightly in primary structure; however, both subgroups are common in that they contain two different dimeric subunits (R1 and R2) and require oxygen in order to form a stable radical. Class Ic RNRs are the most recently discovered, first found in Chlamydia trachomatis. Evidence also suggests its existence in archaea and eubacteria. The sequence of class Ic RNRs shows that residues in the PCET pathway and active site for nucleotide reductase are similar between the three subgroups.
* Class II: Class II RNRs form thiyl radicals with the help of adenosylcobalamin – which fulfills the role of the R2 subunit as a radical generator – and utilize thioredoxin or glutaredoxin as electron donors. Therefore, class II RNRs are made up of only one subunit and present as monomers or dimmers and neither require nor are inhibited by the presence of oxygen.
* Class III: Class III RNRs, like Class I RNRs, are made up of two dimeric protein subunits (NrdG and NrdD); however, unlike in Class I RNRs which require R2 continuously to generate radicals, the small NrdG is only required during the activation of NrdD. The mechanism of Class III RNRs uses formate as an electron donor and generates an oxygen-sensitive glycyl radical, thus rendering the enzymes inactive in the presence of oxygen.

## Radical Mechanism of RNR

Despite the differences in structure and electron donor, all three classes of RNR proceed via a free radical mechanism. Ultimately RNR catalyzes a reaction which results in the replacement of the 2'-hydroxyl group of the ribose with a hydrogen atom resulting in a deoxyribose moiety.

## Metallocofactor Assembly in Class I RNR

Although the Class I RNR’s (Ia, Ib, and Ic) have comparable structures and pathways, the metallocofactors necessarily involved in the activity of RNRs to catalyze the conversion of nucleotides to deoxynucleotides differ remarkably. The mechanisms which generate these cofactors, both in vitro and in vivo, and examining how damaged cofactors are repaired show the significance of each subgroup’s dependence on different cofactors. Studies of the pathways and activation of these metallocofactors have helped our understanding of how biology prevents mismetallation from occurring and configures cluster formation in high yields. All three class I RNR share a common catalytic mechanism in which the metal cofactor is involved directly or indirectly in the oxidation of the conserved cysteine in the active site of alpha to thiol radical S•). Class I RNR oxidation occurs by the Y• in Ia and Ib.
* Class IA: Class IA RNR requires a FeIIIFeIII-Y• cofactor. It is localized in β2 at the end of a hydrophobic channel, the supposed access route for O2 cluster assembly. In studies of E. coli, the in vivo process showed that incubation of apo-β2 of E. coli with FeII, O2, and reductant, resulted in self-assembly of the FeIIIFeIII-Y• cofactor. This process likely requires at minimum a single small protein or molecule to deliver FeII to apo-β2 and to deliver the extra reducing equivalent required to reduce O2 to H2O. This is also plausible because Ia RNRN binds MnII more tightly than FeII, thus requiring some type of chaperone protein to ensure proper metallation.
* Class IB: Class IB RNR is active with both FeIIIFeIII-Y• and MnIIIMnIII-Y• cofactors. The enzymes can form active FeIIIFeIII-Y• cofactors in vitro, but only the MnIIIMnIII-Y• cofactor was found to be relevant in vivo. The mechanism of this formation has been proposed to occur via oxidation of a MnIIMnII center by a flavoprotein known as NrdI, an oxidant created by reduction of O2. In E.Coli, studies have found that the manganese cofactor is induced when iron is at premature levels in the cell, pointing to the significance of manganese in this and other organisms. There is also an extent of organism-dependent variation in metal homeo-stasis to be considered which may help explain why some organisms rely on either cofactor more frequently.
* Class IC: Class IC RNR is unique from Class Ia and Ib RNRs due to its proposed bimetallocofactor, MnIVFeIII. The class Ic RNRs store a one-electron oxidizing equivalent in its metal cluster. In vitro self-assembly of Ic is similar to Ia and Ib in that it reacts with O2 and a reductant to form its respective MnIVFeIII cofactor; however, it differs in that it can also react with 2 equivalents of H2 O2 to form the active cofactor. The class Ic RNR has been isolated from its native organism in vivo, complicating its assembly as the two different metals have similar affinities for the protein. In vitro studies in C. trachomatis have shown the necessity of regulating levels of the metals, along with the order of addition.
There exists problems with proper metal loading within the three subunits of Class I RNR. In the class Ia RNR, it requires a FeIIIFeIII-Y• cofactor, but the protein tends to bind MnII more tightly than FeII. In e.coli, correct metallation of NrdB relies on the necessity of free MnII and FeII present, while iron chaperones are also present to overcome the preference to bind MnII. The issue in class Ib RNR is that it may bind to either FeIIIFeIII-Y• and MnIIIMnIII-Y• cofactors, but only the manganese cofactor was found to be relevant in vivo. Ib binding is dependent on the preference of individual organisms and the concentrations of each metal that they possess inherently. The class Ic RNR complicates metallocofactor assembly since it requires two different metals with similar affinities for the same protein. Regulation of both levels of the metal is important in order to prevent mismetallation and its success depends on the presence of both types of metals. In C. trachomatis, the absence of MnII or at a lower than required rate may lead to diiron cluster formation instead. Thus if these levels are not regulation, low activity and improper metallation occurs. In general, if there is trouble regulating the levels of any of the required metals in each class I RNR, this leads to low activity and improper metallation and ultimately DNA synthesis is affected.

## Biosynthesis and Repair of Metal Cofactors in Class I RNR

Certain general principles and challenges exist when studying the metllocofactor formation with different metals and levels of complexity, as summarized below. Physiological expression conditions are taken into account in studies of metalloenzymes to confirm if the form of protein studied in vitro is the same as its active form in vivo. Class I RNRs can control the concentration of the active metal cofactors through biosynthetic and repeair pathways.
* Cofactors of metal proteins are generated by specific biosynthetic pathways.
* The proteins involved in the biosynthetic pathway are often associated with the operon of the metalloprotein of interest, and certain factors can be analyzed by comparing genomic sequences.
* To facilitate the exchange of ligands and protein factors, metals are transferred in their reduced state.
* There exists a variety of protein factors which include: metal insertase or chaperone to deliver the metal to the active site, specific redox proteins which control the oxidation state of the metal, and GTPases or ATPases which aid in the folding and unfolding processes to allow the metal to be inserted in the active site.
* Due to biological redundancy that affect pathway factors, multiple deletions of genes are required in order to identify phenotypes within a gene deletion experiment.
* A hierarchy of metal delivery to proteins and its regulation is inferred but not completely understood.
* Compartmentalization (e.g. periplasm vs cytosol in prokaryotes) and affinities of proteins to bind certain metals preferentially are two likely factors that contribute to prevent mismetatallion at the cellular level.
* Several proteins have not been isolated from their native source and form heterologous expression systems and leading to mismetallation. Since the optimum level of activity is not fully known, incorrect clusters corresponding to low activity may not be recognized.
* Certain oxidants can cause damage to the metal clusters (e.g. NO and O2) and specific pathways are used in their repair.
* During changes of oxidaion states, protons are typically required for this metal oxidation. Ligands to metal binding can reorganize easily and rearrangement of the carboxylate ligands are critical to the cluster assembly process.
One of the biggest complications is that the metal required for activity is often not the metal that has the highest affinity for binding to a specific protein. The Irving-Williams series (MnII < FeII < CoII < NiII < CuII > ZnII) best describes the relative affinities of proteins for divalent metals, in addition to the dependence on the particular protein coordination environment where the binding takes place. For the latter metals in the series, chaperone proteins exist to aid their movement to the active sites, while intracellularly they are likely to exist as "free" metals at a low concentration. These chaperone proteins also have another function beside delivery, which is to help maintain low levels of free concentration of these metals to prevent mismetallation and binding between other proteins that require MnII and FeII. Compartmentalization can overcome a protein's binding preference, as certain activities occur in different parts of the cell which have and require varying amounts of a metal. In cyanobacteria, it was found that MnII dependent perisplasmic protein must fold in the cytosol where MnII exists freely in a higher amount than ZuII, CuI, and CuII.

## Techniques to Study RNR Activity

There are several techniques used in the laboratory that are used to monitor the activity of the RNR metallocofactors. This contributes to identifying accurate proposed mechanism, generation, and function of these cofactors in vitro and in vivo by studying their movement.
* Whole-Cell Electron Paramagnetic Resonance: EPR was used in studying FeIIIFeIII-Y• biosynthesis in S. cerevisae. It was found that Y• levels were sufficiently high and detectable at endogenous levels in various growth conditions, meaning that the Y• is not modulated as a function of the cell cycle. A small molecule or protein factor must be needed to rapidly reduce the Y• in cell lysates, indicating the presence of a metallocofactor which was later identified to be iron.
* Mossbauer Spectroscopy: This type of spectroscopy monitors iron movement from oxidized and reduced iron pools into the RNR cofactor. It allows for the detection of all oxidation states of iron simultaneously and is sensitive to the surrounding electronic environments of the iron species present. In order for this technique to be accurate, cells first need to be labelled with the Fe57 isotope.
* ↑ Herrick J, Sclavi B. (2007) Ribonucleotide reductase and the regulation of DNA replication: an old story and an ancient heritage Mol Microbiol. 63:22–34
* ↑ Nordlund P, Reichard P (2006). Ribonucleotide Reductases Annu Rev Biochem, 75:681–706
* ↑ Cotruvo, Joseph, Jr., and Stubbe, JoAnne. (2011). Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo Annual Review of Biochemistry, 80: 733-767
* ↑ Eklund H, Eriksson M, Uhlin U, Nordlund P, Logan D (1997). Ribonucleotide reductase--structural studies of a radical enzyme Biol Chem. 378:821–825
* ↑ Cotruvo, Joseph, Jr., and Stubbe, JoAnne. (2011). Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo Annual Review of Biochemistry, 80: 733-767
* ↑ Cotruvo, Joseph, Jr., and Stubbe, JoAnne. (2011). Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo Annual Review of Biochemistry, 80: 733-767
* ↑ Cotruvo, Joseph, Jr., and Stubbe, JoAnne. (2011). Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair In Vitro and In Vivo Annual Review of Biochemistry, 80: 733-767