Radical Enzymes


Radical enzymes harbour a free radical in their polypeptide chain, which participates in catalysis. A growing number of enzymes are known to require a posttranslationally generated free radical for their proper functioning. The radical is generally used to remove a hydrogen atom from an unreactive position in the substrate, activating the substrate to undergo difficult chemistry. Some radical enzymes have unexpectedly been found to harbour a stable radical on an amino acid side‐chain that is distinct from the side‐chains of the active site region; during catalysis, the stable free radical interacts with the active site via radical transfer, and between turnovers it serves as a radical sink.

Key Concepts

  • Radical enzymes utilise single‐electron chemistry to enable difficult chemical reactions.
  • Tyrosyl and tryptophanyl radicals are involved in a large number of biochemical reactions as initiators of catalysis or as partners in radical transfer pathways.
  • Glycyl radicals are oxygen‐sensitive and have only been found in anaerobically expressed enzymes.
  • Transient cysteinyl radicals are important intermediates in a number of radical enzymes.
  • A transient 5′‐deoxyadensyl radical can be formed either by reductive cleavage of the S‐adenosylmethionine cofactor in a radical SAM enzyme or by homolytic cleavage of the vitamin B12 coenzyme AdoCbl.
  • All members of the ribonucleotide reductase enzyme family utilise a transient cysteinyl radical generated by a stable radical in a separate subunit (class I), cleavage of AdoCbl (class II), or a stable glycyl radical (class III).

Keywords: tyrosyl radical; dopa radical; glycyl radical; tryptophanyl radical; cysteinyl radical; 5′‐deoxyadenosyl radical; ribonucleotide reductase

Figure 1. (a) General reaction mechanism of RNRs. (b) General structures of the three RNR classes. (c) Initiator radicals, and intermediates involved in radical transfer and activation. Class I RNR consists of two homodimeric subunits, the catalytic dimer contains the active site and the radical‐containing dimer the stable radical. Class II RNR is monomeric (shown here) or homodimeric (not shown) and harbour an AdoCbl cofactor close to the active site. Class III RNRs are homodimeric with a stable glycyl radical close to the active site; once the glycyl radical is introduced by NrdG, a specific activating enzyme (a radical‐SAM enzyme), the class III RNR can perform multiple turnovers. The catalytic core structure in all three RNR classes is a specific 10‐stranded β/α‐barrel, which also occurs in all glycyl radical enzymes (GREs; see below). Structures used: class I, (human R1: 2WGH, E. coli R2: 1MXR); class II, L. leichmannii monomeric protein (1L1L); class III, bacteriophage T4 (1H7A). The β‐barrel and catalytic radical cysteine (left protomer for dimeric proteins) are oriented in a similar orientation.
Figure 2. (a) Radical transfer pathway in the class Ia holoenzyme (left), and general structure of the R2 protein (right). Structures used: E. coli class Ia R1–R2 complex, (6W4X); E. coli class Ia R2, (1MXR). (b) Structure of the stable radical in the R2 subclasses Ia to Ie. Structures used: Ia, E. coli (1MXR); Ib, C. ammoniagenes (1KGP); Ic, Chlamydia trachomatis (1SYY, 4D8G); Id, Leeuwenhoekiella blandensis (6SF5); Ie, Mesoplasma florum (6GP2). Solvent derived ligands are omitted for clarity. The location of the radical species is indicated in green.
Figure 3. Prostaglandin H synthase. (a) Structure of the active site region and (b) proposed radical reaction mechanism (structure and numbering refer to PGHS‐1 from Ovis aries: 1Q4G). The activation of AA is initiated by site‐selective H‐atom abstraction by a Y· radical. The haem is shown in yellow, and the aspirin analogue 2‐bromoacetoxybenzoic acid and the aspirin antagonist salicylic acid in red. AA, arachidonic acid; PP, protoporphyrin, PGG2, prostaglandin G2; PGH2, prostaglandin H2.
Figure 4. Schematic structure of Photosystem II and the catalytic cycle of the OEC (the Kok cycle). (a) Selected redox‐active components highlighted, including the OEC (CaMn4) and the two tyrosyl radical residues (YD and YD). Also shown is the chromophore P680 and key electron mediators Pheophytin (Pheo) and Quinones A and B. (b) A schematic representation of the Kok cycle, successive light flashes cycles the OEC through four metastable intermediates S0–S3 and the final light flash results in O2 evolution proceeding via the putative S4 state. The primary electron acceptor of the OEC is a tyrosyl radical (Yz) continuously regenerated via oxidation by P680.
Figure 5. The active site core structure of GREs where the Gly‐loop and Cys‐loop meet in a 10‐stranded β/α barrel. Structure used: Bacteriophage T4 class III RNR (1H7A).
Figure 6. Activation, oxygen inactivation, and YfiD‐mediated reactivation of PFL. PFL (blue) with Gly‐loop (red); PFL activating enzyme (green), YfiD (brown). PFL activating enzyme promotes a conformational change of Gly‐loop during activation (1), whereafter active PFL with Gly· is generated (2). Upon oxygen exposure, PFL is irreversibly cleaved at the Gly· site (3), and the C‐terminal PFL peptide is ejected (4). YfiD binds exposed to PFL and is activated by PFL activating enzyme when oxygen has disappeared (5), whereafter Gly· in YfiD occupies the Gly‐loop site in PFL (6).
Figure 7. Schematic comparison of how the 5′‐deoxyadenosyl radical (5′‐dAdo·) is formed from the ‘omega’ intermediate (an organometallic complex of the 5′‐C of the AdoMet‐derived deoxyadenosyl moiety bound to the AdoMet‐coordinating Fe in the [4Fe‐4S] cluster) derived from the radical SAM cofactor (only sulfur of Met residue shown for increased clarity) observed in radical SAM enzymes and from the AdoCbI cofactor (Table ).
Figure 8. The typical TIM barrel in a majority of radical SAM enzymes with AdoMet bound to the [4Fe‐4S] cluster. Structure used: Methanosarcina barkeri str. Fusaro methylornithine synthase (3T7V).


Andersson J, Westman M, Sahlin M and Sjöberg B‐M (2000) Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD. The Journal of Biological Chemistry 275: 19449–19455.

Aurelius O, Johansson R, Bagenholm V, et al. (2015) The crystal structure of thermotoga maritima class III ribonucleotide reductase lacks a radical cysteine pre‐positioned in the active site. PLoS One 10: e0128199.

Backman LRF, Funk MA, Dawson CD and Drennan CL (2017) New tricks for the glycyl radical enzyme family. Critical Reviews in Biochemistry and Molecular Biology 52: 674–695.

Backman LR, Huang YY, Andorfer MC, et al. (2020) Molecular basis for catabolism of the abundant metabolite trans‐4‐hydroxy‐L‐proline by a microbial glycyl radical enzyme. eLife 9.

Barry BA and Babcock GT (1988) Characterization of the tyrosine radical involved in photosynthetic oxygen evolution. Chemica Scripta 28A: 117–122.

Blaesi EJ, Palowitch GM, Hu K, et al. (2018) Metal‐free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine‐derived dihydroxyphenylalanine radical. Proceedings of the National Academy of Sciences of the United States of America 115: 10022–10027.

Bleifuss G, Kolberg M, Pötsch S, et al. (2001) Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high‐field EPR study at 94 GHz. Biochemistry 40: 15362–15368.

Bodea S, Funk MA, Balskus EP and Drennan CL (2016) Molecular Basis of C‐N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine‐Lyase. Cell Chem Biol 23: 1206–1216.

Bourdon A, Minai L, Serre V, et al. (2007) Mutation of RRM2B, encoding p53‐controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nature Genetics 39: 776–780.

Bowman SEJ, Backman LRF, Bjork RE, et al. (2019) Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen‐damaged glycyl radical enzyme. Journal of Biological Inorganic Chemistry 24: 817–829.

Broderick JB, Duffus BR, Duschene KS and Shepard EM (2014) Radical S‐adenosylmethionine enzymes. Chemical Reviews 114: 4229–4317.

Buckel W and Golding BT (2006) Radical enzymes in anaerobes. Annual Review of Microbiology 60: 27–49.

Byer AS, Yang H, McDaniel EC, et al. (2018) Paradigm shift for radical S‐adenosyl‐l‐methionine reactions: the organometallic intermediate omega is central to catalysis. Journal of the American Chemical Society 140: 8634–8638.

van Dam PJ, Willems J‐P, Schmidt PP, et al. (1998) High‐frequency EPR and pulsed Q‐band ENDOR studies on the origin of the hydrogen bond in tyrosyl radicals of ribonucleotide reductase R2 proteins from mouse and herpes simplex virus type 1. Journal of the American Chemical Society 120: 5080–5085.

Dodson CA, Hore PJ and Wallace MI (2013) A radical sense of direction: signalling and mechanism in cryptochrome magnetoreception. Trends in Biochemical Sciences 38: 435–446.

Ferlez B, Sutter M and Kerfeld CA (2019) Glycyl radical enzyme‐associated microcompartments: redox‐replete bacterial organelles. MBio: 10.

Funk MA, Judd ET, Marsh EN, Elliott SJ and Drennan CL (2014) Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity. Proceedings of the National Academy of Sciences of the United States of America 111: 10161–10166.

Goulah CC, Zhu G, Koszelak‐Rosenblum M and Malkowski MG (2013) The crystal structure of alpha‐dioxygenase provides insight into diversity in the cyclooxygenase‐peroxidase superfamily. Biochemistry 52: 1364–1372.

Guittet O, Håkansson P, Voevodskaya N, et al. (2001) Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells. The Journal of Biological Chemistry 276: 40647–40651.

Heider J, Szaleniec M, Martins BM, et al. (2016) Structure and function of benzylsuccinate synthase and related fumarate‐adding glycyl radical enzymes. Journal of Molecular Microbiology and Biotechnology 26: 29–44.

Himo F, Gräslund A and Eriksson LA (1997) Density functional calculations on model tyrosyl radicals. Biophysical Journal 72: 1556–1567.

Holliday GL, Akiva E, Meng EC, et al. (2018) Atlas of the radical SAM superfamily: divergent evolution of function using a “plug and play” domain. Methods in Enzymology 606: 1–71.

Horitani M, Shisler K, Broderick WE, et al. (2016) Radical SAM catalysis via an organometallic intermediate with an Fe‐[5'‐C]‐deoxyadenosyl bond. Science 352: 822–825.

Huff GS, Doncheva IS, Brinkley DW, et al. (2011) Experimental and computational investigations of oxygen reactivity in a heme and tyrosyl radical‐containing fatty acid alpha‐(di)oxygenase. Biochemistry 50: 7375–7389.

Huyett JE, Doan PE, Gurbiel R, et al. (1995) Compound ES of cytochrome c peroxidase contains a Trp .pi.‐cation radical: characterization by continuous wave and pulsed Q‐band external nuclear double resonance spectroscopy. Journal of the American Chemical Society 117: 9033–9041.

Högbom M, Galander M, Andersson M, et al. (2003) Displacement of the tyrosyl radical cofactor in ribonucleotide reductase obtained by single‐crystal high‐field EPR and 1.4‐A x‐ray data. Proceedings of the National Academy of Sciences of the United States of America 100: 3209–3214.

Högbom M (2011) Metal use in ribonucleotide reductase R2, di‐iron, di‐manganese and heterodinuclear‐‐an intricate bioinorganic workaround to use different metals for the same reaction. Metallomics 3: 110–120.

Kang G, Taguchi AT, Stubbe J and Drennan CL (2020) Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex. Science 368: 424–427.

Kern J, Chatterjee R, Young ID, et al. (2018) Structures of the intermediates of Kok's photosynthetic water oxidation clock. Nature 563: 421–425.

Kovacevic B, Baric D, Babic D, et al. (2018) Computational tale of two enzymes: glycerol dehydration with or without B12. Journal of the American Chemical Society 140: 8487–8496.

Lendzian F, Sahlin M, Macmillan F, et al. (1996) Electronic structure of neutral tryptophan radicals in ribonucleotide reductase studied by EPR and ENDOR spectroscopy. Journal of the American Chemical Society 118: 8111–8120.

Levin BJ and Balskus EP (2018) Characterization of 1,2‐propanediol dehydratases reveals distinct mechanisms for B12‐dependent and glycyl radical enzymes. Biochemistry 57: 3222–3226.

Licht S, Gerfen GJ and Stubbe JA (1996) Thiyl radicals in ribonucleotide reductases. Science 271: 477–481.

Licht SS, Booker S and Stubbe J (1999) Studies on the catalysis of carbon‐cobalt bond homolysis by ribonucleoside triphosphate reductase: evidence for concerted carbon‐cobalt bond homolysis and thiyl radical formation. Biochemistry 38: 1221–1233.

Loderer C, Jonna VR, Crona M, et al. (2017) A unique cysteine‐rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover. The Journal of Biological Chemistry 292: 19044–19054.

Logan DT, Mulliez E, Larsson KM, et al. (2003) A metal‐binding site in the catalytic subunit of anaerobic ribonucleotide reductase. Proceedings of the National Academy of Sciences of the United States of America 100: 3826–3831.

Marsh EN, Patterson DP and Li L (2010) Adenosyl radical: reagent and catalyst in enzyme reactions. Chembiochem 11: 604–621.

Mukherjee A, Angeles‐Boza AM, Huff GS and Roth JP (2011) Catalytic mechanism of a heme and tyrosyl radical‐containing fatty acid alpha‐(di)oxygenase. Journal of the American Chemical Society 133: 227–238.

Mulliez E, Padovani D, Atta M, Alcouffe C and Fontecave M (2001) Activation of class III ribonucleotide reductase by flavodoxin: a protein radical‐driven electron transfer to the iron‐sulfur center. Biochemistry 40: 3730–3736.

Nicolet Y (2020) Structure‐function relationships of radical SAM enzymes. Nature Catalysis 3: 337–350.

Peck SC, Denger K, Burrichter A, et al. (2019) A glycyl radical enzyme enables hydrogen sulfide production by the human intestinal bacterium Bilophila wadsworthia. Proceedings of the National Academy of Sciences of the United States of America 116: 3171–3176.

Reichard P (1995) To be there when the picture is painted. Annual Review of Biochemistry 64: 1–28.

Sayler RI, Stich TA, Joshi S, et al. (2019) Trapping and electron paramagnetic resonance characterization of the 5′dAdo(*) radical in a radical S‐adenosyl methionine enzyme reaction with a non‐native substrate. ACS Central Science 5: 1777–1785.

Selvaraj B, Buckel W, Golding BT, Ullmann GM and Martins BM (2016) Structure and function of 4‐hydroxyphenylacetate decarboxylase and its cognate activating enzyme. Journal of Molecular Microbiology and Biotechnology 26: 76–91.

Seo MJ and Oh DK (2017) Prostaglandin synthases: molecular characterization and involvement in prostaglandin biosynthesis. Progress in Lipid Research 66: 50–68.

Shisler KA and Broderick JB (2014) Glycyl radical activating enzymes: structure, mechanism, and substrate interactions. Archives of Biochemistry and Biophysics 546: 64–71.

Sjöberg B‐M, Reichard P, Gräslund A and Ehrenberg A (1978) The tyrosine free radical in ribonucleotide reductase from Escherichia coli. The Journal of Biological Chemistry 253: 6863–6865.

Sofia HJ, Chen G, Hetzler BG, Reyes‐Spindola JF and Miller NE (2001) Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods. Nucleic Acids Research 29: 1097–1106.

Srinivas V, Lebrette H, Lundin D, et al. (2018) Metal‐free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens. Nature 563: 416–420.

Stoll S, Shafaat HS, Krzystek J, et al. (2011) Hydrogen bonding of tryptophan radicals revealed by EPR at 700 GHz. Journal of the American Chemical Society 133: 18098–18101.

Styring S, Sjöholm J and Mamedov F (2012) Two tyrosines that changed the world: Interfacing the oxidizing power of photochemistry to water splitting in photosystem II. Biochim Biophys Acta 1917: 78–87.

Suga M, Akita F, Hirata K, et al. (2015) Native structure of photosystem II at 1.95 A resolution viewed by femtosecond X‐ray pulses. Nature 517: 99–103.

Sun XY, Ollagnier S, Schmidt PP, et al. (1996) The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681. The Journal of Biological Chemistry 271: 6827–6831.

Tamao Y and Blakley RL (1973) Direct spectrophotometric observation of an intermediate formed from deoxyadenosylcobalamin in ribonucleotide reduction. Biochemistry 12: 24–34.

Tsai AL, Hsi LC, Kulmacz RJ, Palmer G and Smith WL (1994) Characterization of the tyrosyl radicals in ovine prostaglandin H synthase‐1 by isotope replacement and site‐directed mutagenesis. The Journal of Biological Chemistry 269: 5085–5091.

Volkov AN, Nicholls P and Worrall JA (2011) The complex of cytochrome c and cytochrome c peroxidase: the end of the road? Biochimica et Biophysica Acta 1807: 1482–1503.

Wagner AF, Frey M, Neugebauer FA, Schäfer W and Knappe J (1992) The free radical in pyruvate formate‐lyase is located on glycine‐734. Proceedings of the National Academy of Sciences of the United States of America 89: 996–1000.

Wagner AF, Schultz S, Bomke J, et al. (2001) YfiD of Escherichia coli and Y06I of bacteriophage T4 as autonomous glycyl radical cofactors reconstituting the catalytic center of oxygen‐fragmented pyruvate formate‐lyase. Biochemical and Biophysical Research Communications 285: 456–462.

Wei Y, Funk MA, Rosado LA, et al. (2014) The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant. Proceedings of the National Academy of Sciences of the United States of America 111: E3756–E3765.

Whittaker MM and Whittaker JW (1990) A tyrosine‐derived free radical in apogalactose oxidase. The Journal of Biological Chemistry 265: 9610–9613.

Wikström M, Krab K and Sharma V (2018) Oxygen activation and energy conservation by cytochrome c oxidase. Chemical Reviews 118: 2469–2490.

Yamamoto J, Shimizu K, Kanda T, et al. (2017) Loss of fourth electron‐transferring tryptophan in animal (6‐4) photolyase impairs DNA repair activity in bacterial cells. Biochemistry 56: 5356–5364.

Yang H, Impano S, Shepard EM, et al. (2019) Photoinduced electron transfer in a radical SAM enzyme generates an S‐adenosylmethionine derived methyl radical. Journal of the American Chemical Society 141: 16117–16124.

Yin DT, Urresti S, Lafond M, et al. (2015) Structure‐function characterization reveals new catalytic diversity in the galactose oxidase and glyoxal oxidase family. Nature Communications 6: 10197.

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Högbom, Martin, Sjöberg, Britt‐Marie, and Berggren, Gustav(Sep 2020) Radical Enzymes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0029205]