Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine.

The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced. Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors. From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined. A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected. ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist. Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern. The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.     

Electron nuclear double resonance studies of radicals produced by the PbO2 oxidation of α-tocopherol and its model compound in solution

The electron nuclear double resonance (ENDOR) spectra of chromanoxyl radicals obtained by the PbO₂ oxidation of α-tocopherol and its model compound were observed in t-butylbenzene, and the proton hyperfine coupling constants were correctly determined. Each of the two β- and γ-methylene protons in the chromanoxyl ring shows an equivalent hyperfine splitting, suggesting that the heterocyclic ring attached to the aromatic ring are coplanar with the plane of the aromatic system. A comparison of the hyperfine couplings in α-tocopheroxyl radical and its model shows that the introduction of a long-isoprenoid-chain in the α-tocopherol in place of a methyl group in the model compound has very little effect on the unpaired spin distribution or molecular structure of the chromanoxyl skeleton. The results of McLachlan molecular orbital (MO) calculations were found to be in satisfactory agreement with the ‘experimental’ spin densities evaluated from the hyperfine coupling constants.     

Electron spin resonance studies of chromanoxyl radicals derived from tocopherols

Electron spin resonance measurements were performed for the chromanoxyl radicals obtained from -, β-, γ-, δ-tocopherol, tocol and their model compounds by oxidizing the phenol precursors with PbO₂ in toluene. The proton hyperfine coupling constants were determined, and the ‘experimental’ spin densities were evaluated from the hyperfine coupling constants. From the results, the methyl-substitution effects on the unpaired spin distribution and molecular structure of the chromanoxyl radicals have been studied.     

Tyrosyl radicals in photosystem II

In PSII, there are two redox-active tyrosines, D and Z, with different midpoint potentials and different reduction kinetics. The factors responsible for these functional differences have not yet been elucidated. Recent model compound studies of tyrosinate and of tyrosine-containing dipeptides have demonstrated that perturbations of the amino and amide/imide group occur when the tyrosyl aromatic ring is oxidized [J. Am. Chem. Soc. 124 (2002) 5496]. Accompanying density functional calculations suggested that this perturbation is due to spin density delocalization from the aromatic ring onto the amino nitrogen. The implication of this finding is that spin density delocalization may occur in redox-active, tyrosine-containing enzymes, like Photosystem II. In this paper, we review the supporting evidence for the hypothesis that tyrosyl radical spin density delocalizes into the peptide bond in a conformationally sensitive, sequence-dependent manner. Our experimental measurements on tyrosyl radicals in dipeptides have suggested that the magnitude of the putative spin migration may be sequence-dependent. Vibrational spectroscopic studies on the tyrosyl radicals in Photosystem II, which are consistent with spin migration, are reviewed. Migration of the unpaired spin may provide a mechanism for control of the direction and possibly the rate of electron transfer.     

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides, which are essential for DNA synthesis and repair in all organisms. The three currently known classes of RNRs are postulated to utilize a similar mechanism for ribonucleotide reduction via a transient thiyl radical, but they differ in the way this radical is generated. Class I RNR, found in all eukaryotic organisms and in some eubacteria and viruses, employs a diferric iron center and a stable tyrosyl radical in a second protein subunit, R2, to drive thiyl radical generation near the substrate binding site in subunit R1. From extensive experimental and theoretical research during the last decades, a general mechanistic model for class I RNR has emerged, showing three major mechanistic steps: generation of the tyrosyl radical by the diiron center in subunit R2, radical transfer to generate the proposed thiyl radical near the substrate bound in subunit R1, and finally catalytic reduction of the bound ribonucleotide. Amino acid- or substrate-derived radicals are involved in all three major reactions. This article summarizes the present mechanistic picture of class I RNR and highlights experimental and theoretical approaches that have contributed to our current understanding of this important class of radical enzymes.     

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.     

A new method of identifying the site of tyrosyl radicals in proteins

Protein-bound tyrosyl radicals catalyze many important enzymatic reactions. They can also initiate oxidative damage to cells. Here we report a new method of computer simulation of tyrosyl radical electron paramagnetic resonance spectra. The method enables the determination of the rotational conformation of the phenoxyl ring in a radical with unprecedented accuracy (approximately 2 degrees ). When coupled with a new online database, all tyrosine residues in a protein can be screened for that particular conformation. For the first time we show relationships between the spin density on atom C1 (rho(C1)) and the principal g-factors measured by electron paramagnetic resonance spectroscopy (rho(C1) on g(x) is shown to be linear). The new method enables the accurate determination of rho(C1) in all known tyrosyl radicals, evaluates the likelihood of a hydrogen bond, and determines the possibility of a rho(C1) distribution in the radicals. This information, together with the accurately determined rotational conformation, is frequently sufficient to allow for an unambiguous identification of the site of radical formation. The possibility of a similar relationship between rho(C) and g(x) in other radicals, e.g., tryptophanyl, is discussed.     

Tyrosine iminoxyl radical formation from tyrosyl radical/nitric oxide and nitrosotyrosine

The quenching of the Y(D)(.) tyrosyl radical in photosystem II by nitric oxide was reported to result from the formation of a weak tyrosyl radical-nitric oxide complex (Petrouleas, V., and Diner, B. A. (1990) Biochim. Biophys. Acta 1015, 131-140). This radical/radical reaction is expected to generate an electron spin resonance (ESR)-silent 3-nitrosocyclohexadienone species that can reversibly regenerate the tyrosyl radical and nitric oxide or undergo rearrangement to form 3-nitrosotyrosine. It has been proposed that 3-nitrosotyrosine can be oxidized by one electron to form the tyrosine iminoxyl radical (>C=N-O*). This proposal was put forth as a result of ESR detection of the iminoxyl radical intermediate when photosystem II was exposed to nitric oxide (Sanakis, Y., Goussias, C., Mason, R. P., and Petrouleas, V. (1997) Biochemistry 36, 1411-1417). A similar iminoxyl radical was detected in prostaglandin H synthase-2 (Gunther, M. R., Hsi, L. C., Curtis, J. F., Gierse, J. K., Marnett, L. J., Eling, T. E., and Mason, R. P. (1997) J. Biol. Chem., 272, 17086-17090). Although the iminoxyl radicals detected in the photosystem II and prostaglandin H synthase-2 systems strongly suggest a mechanism involving 3-nitrosotyrosine, the iminoxyl radical ESR spectrum was not unequivocally identified as originating from tyrosine. We report here the detection of the non-protein L-tyrosine iminoxyl radical generated by two methods: 1) peroxidase oxidation of synthetic 3-nitroso-N-acetyl-L-tyrosine and 2) peroxidase oxidation of free L-tyrosine in the presence of nitric oxide. A newly developed ESR technique that uses immobilized enzyme was used to perform the ESR experiments. Analysis of the high resolution ESR spectrum of the tyrosine iminoxyl radical generated from free tyrosine and nitric oxide reveals a 28.4-G isotropic nitrogen hyperfine coupling and a 2.2-G proton hyperfine coupling assigned to the proton originally ortho to the phenoxyl oxygen.     

Generation and electron paramagnetic resonance spin trapping detection of thiyl radicals in model proteins and in the R1 subunit of Escherichia coli ribonucleotide reductase.

In the Escherichia coli class Ia ribonucleotide reductase (RNR), the best characterized RNR, there is no spectroscopic evidence for the existence of the postulated catalytically essential thiyl radical (R-S(*)) in the substrate binding subunit R1. We report first results on artificially generated thiyl radicals in R1 using two different methods: chemical oxidation by Ce(IV)/nitrilotriacetate (NTA) and laser photolysis of nitric oxide from nitrosylated cysteines. In both cases, EPR spin trapping at room temperature using phenyl-N-t-butylnitrone, and controls with chemically blocked cysteines, has shown that the observed spin adduct originates from thiyl radicals. The EPR line shape of the protein-bound spin adduct is typical for slow motion of the nitroxide moiety, which indicates that the majority of trapped thiyl radicals are localized in a folded region of R1. In aerobic R1 samples without spin trap that were frozen after treatment with Ce(IV)/NTA or laser photolysis, we observed sulfinyl radicals (R-S(*)=O) assigned via their g-tensor components 2.0213, 2.0094, and 2.0018 and the hyperfine tensor components 1.0, 1.1, and 0.9 mT of one beta-proton. Sulfinyl radicals are the reaction products of thiyl radicals and oxygen and give additional evidence for generation of thiyl radicals in R1 by the procedures used.     

Structure of the high-valent FeIIIFeIV state in ribonucleotide reductase (RNR) of Chlamydia trachomatis--combined EPR, 57Fe-, 1H-ENDOR and X-ray studies

A recently discovered subgroup of class I ribonucleotide reductase (RNR) found in the infectious bacterium Chlamydia trachomatis (C. trachomatis) was shown to exhibit a high-valent Fe(III)Fe(IV) center instead of the tyrosyl radical observed normally in all class I RNRs. The X-ray structure showed that C. trachomatis WT RNR has a phenylalanine at the position of the active tyrosine in Escherichia coli RNR. In this paper the X-ray structure of variant F127Y is presented, where the tyrosine is restored. Using (1)H- and (57)Fe-ENDOR spectroscopy it is shown, that in WT and variants F127Y and Y129F of C. trachomatis RNR, the Fe(III)Fe(IV) center is virtually identical with the short-lived intermediate X observed during the iron oxygen reconstitution reaction in class I RNR from E. coli. The experimental data are consistent with a recent theoretical model for X, proposing two bridging oxo ligands and one terminal water ligand. A surprising extension of the lifetime of the Fe(III)Fe(IV) state in C. trachomatis from a few seconds to several hours at room temperature was observed under catalytic conditions in the presence of substrate. These findings suggest a possible new role for the Fe(III)Fe(IV) state also in other class I RNR, during the catalytic radical transfer reaction, by which the substrate turnover is started.     

Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.     

Stopped-flow investigation of antioxidant activity of tocopherols. Finding of new tocopherol derivatives having higher antioxidant activity than alpha-tocopherol

Second-order rate constants, kappa s, for H-atom abstraction by phenoxyl radicals from five tocopherol (vitamin E) derivatives have been measured spectrophotometrically at 25.0 degrees C by the stopped-flow method, as a model reaction of tocopherols with unstable free radicals (LOO., LO., and HO.) in biological systems. Three new tocopherol derivatives with a five-membered heterocyclic ring were found to be 1.9-2.1 times more active than the alpha-tocopherol which has the highest antioxidant activity among natural tocopherols. The proton hyperfine splittings for the five tocopheroxyl radicals derived from these tocopherols by the reaction with phenoxyl were also determined by ESR measurements.     

Studies of ribonucleotide reductase in crucian carp-an oxygen dependent enzyme in an anoxia tolerant vertebrate

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O(2)) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp.     

Ribonucleotide reductase modularity: Atypical duplication of the ATP-cone domain in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa, which causes serious nosocomial infections, is a gamma-proteobacterium that can live in many different environments. Interestingly P. aeruginosa encodes three ribonucleotide reductases (RNRs) that all differ from other well known RNRs. The RNR enzymes are central for de novo synthesis of deoxyribonucleotides and essential to all living cells. The RNR of this study (class Ia) is a complex of the NrdA protein harboring the active site and the allosteric sites and the NrdB protein harboring a tyrosyl radical necessary to initiate catalysis. P. aeruginosa NrdA contains an atypical duplication of the N-terminal ATP-cone, an allosteric domain that can bind either ATP or dATP and regulates the overall enzyme activity. Here we characterized the wild type NrdA and two truncated NrdA variants with precise N-terminal deletions. The N-terminal ATP-cone (ATP-c1) is allosterically functional, whereas the internal ATP-cone lacks allosteric activity. The P. aeruginosa NrdB is also atypical with an unusually short lived tyrosyl radical, which is efficiently regenerated in presence of oxygen as the iron ions remain tightly bound to the protein. The P. aeruginosa wild type NrdA and NrdB proteins form an extraordinarily tight complex with a suggested alpha4beta4 composition. An alpha2beta2 composition is suggested for the complex of truncated NrdA (lacking ATP-c1) and wild type NrdB. Duplication or triplication of the ATP-cone is found in some other bacterial class Ia RNRs. We suggest that protein modularity built on the common catalytic core of all RNRs plays an important role in class diversification within the RNR family.     

Control of metallation and active cofactor assembly in the class Ia and Ib ribonucleotide reductases: diiron or dimanganese?

Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms. Activity of the class Ia and Ib RNRs requires a stable tyrosyl radical (Y?), which can be generated by the reaction of O2 with a diferrous cluster on the β subunit to form active diferric-Y? cofactor. Recent experiments have demonstrated, however, that in vivo the class Ib RNR contains an active dimanganese(III)-Y? cofactor. The similar metal binding sites of the class Ia and Ib RNRs, their ability to bind both MnII and FeII, and the activity of the class Ib RNR with both diferric-Y? and dimanganese(III)-Y cofactors raise the intriguing question of how the cell prevents mismetallation of these essential enzymes. The presence of the class Ib RNR in numerous pathogenic bacteria also highlights the importance of manganese for these organisms' growth and virulence.     

Redox state dependence of rotamer distributions in tyrosine and neutral tyrosyl radical

Redox state-dependent changes in the relative orientation of the phenol side chain and the peptide group in model tyrosine have been characterized using specific 2H isotopic labelling and X-band electron paramagnetic resonance (EPR) spectroscopy. Tyrosyl radicals were generated by UV photolysis of tyrosine trapped in rigid polycrystalline basic-aqueous medium at T < or = 170 K. Ring-2H(4) and beta-2H(2) substitutions on tyrosine were used to enhance the lineshape contributions from beta-hydrogen or ring-hydrogen hyperfine interactions, respectively. The EPR lineshape at 120 K of the trapped ring-2H(4)-tyrosyl radical is altered dramatically after annealing at 235 K. In contrast, the lineshape of the beta-2H(2)-tyrosyl radical is impervious to annealing. The effect of annealing on the lineshape therefore arises from a change in the isotropic hyperfine coupling between unpaired pi-electron spin density at the ring carbon atom C(1) and the beta-hydrogen nuclei, which is caused by rotational relaxation of the ring and peptide group about the C(1)-C(beta) bond. EPR simulations indicate angular distributions of the peptide group (R-) of 0 degrees < or = theta(R) < or = 30 degrees and 0 degrees < or = theta(R)< or = 18 degrees in the rigid and relaxed radical states, respectively. Redox-induced changes in the C(1)-C(beta) rotamer distribution must be accounted for in assessments of stable amino acid side chain equilibrium structures, and may influence catalytic tyrosyl radical/tyrosine function in enzymes.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.     

Electron spin resonance and pulse radiolysis studies on the spin trapping of sulphur-centered radicals

Thiyl radicals are shown to be readily trapped with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (TMPO) giving characteristic spin adducts with hyperfine coupling constants aN 1.52-1.58, aH 1.52-1.80 mT, and g values in the range 2.0065-2.0067 for the DMPO adducts and aN 1.50-1.56, aH 1.70-1.92 mT, g 20049-2.0051 for the TMPO adducts. Kinetic data obtained from pulse radiolysis studies show that, in general, thiyl radicals react rapidly with these spin traps with rate constants of the order of 10(7)-10(8) dm3 mol-1 s-1. The tetramethylated spin trap TMPO though giving slightly less intense electron spin resonance (ESR) spectra, produces longer lived adducts, and is suggested to be of greater utility due to the more characteristic nature of the coupling constants of the observed adducts; reaction of certain thiyl radicals with DMPO produces adducts which are superficially similar to the hydroxyl radical adduct to the same trap.     

Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy.

Structural changes in Escherichia coli DNA photolyase induced by binding of a (cis,syn)-cyclobutane pyrimidine dimer (CPD) are studied by continuous-wave electron paramagnetic resonance and electron-nuclear double resonance spectroscopies, using the flavin adenine dinucleotide (FAD) cofactor in its neutral radical form as a naturally occurring electron spin probe. The electron paramagnetic resonance/electron-nuclear double resonance spectral changes are consistent with a large distance (> or =0.6 nm) between the CPD lesion and the 7,8-dimethyl isoalloxazine ring of FAD, as was predicted by recent model calculations on photolyase enzyme-substrate complexes. Small shifts of the isotropic proton hyperfine coupling constants within the FAD's isoalloxazine moiety can be understood in terms of the cofactor binding site becoming more nonpolar because of the displacement of water molecules upon CPD docking to the enzyme. Molecular orbital calculations of hyperfine couplings using density functional theory, in conjunction with an isodensity polarized continuum model, are presented to rationalize these shifts in terms of the changed polarity of the medium surrounding the FAD cofactor.     

The periodic table of ribonucleotide reductases

In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2′-deoxynucleotide building blocks of DNA. The metal ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, β, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.