A Mycobacterium tuberculosis ligand-binding Mn/Feprotein reveals a new cofactor in a remodeledR2-protein scaffoldCharlotta S. Anderssona and Martin Högboma,b,1

aStockholm Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for NaturalSciences C4, SE-106 91 Stockholm, Sweden; and bDepartment of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596,SE-751 24 Uppsala, Sweden

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved February 23, 2009 (received for review December 20, 2008)

Chlamydia trachomatis R2c is the prototype for a recently discov-ered group of ribonucleotide reductase R2 proteins that use aheterodinuclear Mn/Fe redox cofactor for radical generation andstorage. Here, we show that the Mycobacterium tuberculosisprotein Rv0233, an R2 homologue and a potential virulence factor,contains the heterodinuclear manganese/iron-carboxylate cofac-tor but displays a drastic remodeling of the R2 protein scaffold intoa ligand-binding oxidase. The first structural characterization ofthe heterodinuclear cofactor shows that the site is highly specificfor manganese and iron in their respective positions despite asymmetric arrangement of coordinating residues. In this proteinscaffold, the Mn/Fe cofactor supports potent 2-electron oxidationsas revealed by an unprecedented tyrosine-valine crosslink in theactive site. This wolf in sheep’s clothing defines a distinct func-tional group among R2 homologues and may represent a structuraland functional counterpart of the evolutionary ancestor of R2s andbacterial multicomponent monooxygenases.

bioinorganic chemistry � diiron � manganese � monooxygenase � R2c

D iiron-carboxylate proteins perform some of the most chemi-cally challenging oxidations observed in nature. The 2 beststudied groups are the bacterial multicomponent monooxygenases(BMMs) and the ribonucleotide reductase R2 proteins. BMMs usethe diiron cofactor to perform a 2-electron oxidation, the O2-dependent hydroxylation of hydrocarbons, including the hydroxy-lation of methane to methanol performed by soluble methanemonooxygenase (MMO) via a Fe(IV)-Fe(IV) intermediate (1–3).BMMs have different and usually broad substrate specificities,including alkanes, alkenes, and aromatic compounds. For thisreason the proteins and the bacteria that produce them are of greatinterest for industrial and environmental applications, such asbioremediation of contaminated soil. All diiron hydrocarbon hy-droxylases/monooxygenases are multisubunit complexes requiringdifferent protein components for activity, complicating their use inbiotechnological applications (4–6).

Ribonucleotide reductases (RNRs) are the only identified en-zymes for de novo synthesis of all four deoxyribonucleotides. TheR2 subunit of Class-I RNRs is a homodimeric diiron-carboxylateprotein that performs a 1-electron oxidation. Standard R2s gener-ate an essential stable tyrosyl radical (Y�) via a Fe(III)-Fe(IV)intermediate (7–10).

Diiron-carboxylate proteins have very similar metal sites, coor-dinated by 4 carboxylates and 2 histidines, and are believed to sharea common evolutionary ancestor (6, 11). Much effort has gone intodefining the structural and chemical determinants that direct thesystems to perform 1- or 2-electron chemistry (4, 12, 13). A peculiarR2, lacking the radical harboring tyrosine, was identified in theimportant human pathogen Chlamydia trachomatis (14, 15). It wassuggested that the 1-electron oxidizing equivalent was stored at themetal site, as opposed to as a tyrosyl radical, and that this could bean adaptation to produce a radical site that is less sensitive toscavenging by reactive nitrogen and oxygen species produced by the

host’s immune response. In addition, a number of proteins, previ-ously assigned as standard R2s, were assigned to this group ofChlamydia R2-like proteins, denoted R2c. Recently it was shownthat CtR2c possesses a manganese/iron redox cofactor and that theone-electron oxidizing equivalent is stored as a Mn(IV)-Fe(III)species that replaces the Fe(III)-Fe(III)-Y� cofactor in standard R2s(16, 17). Interestingly, it was also shown that not only is theMn(IV)-Fe(III) form stable to incubations with H2O2, but thereduced forms are efficiently activated by H2O2 treatment (18).

The Mycobacterium tuberculosis R2c-like protein, Rv0233, is 1 ofthe 10 most up-regulated proteins, about 7-fold, in the virulentH37Rv M. tuberculosis strain compared with the avirulent bacillusCalmette–Guérin (BCG) vaccine strain and is therefore a possiblevirulence factor and drug target candidate (19). M. tuberculosis isthe causative agent of tuberculosis (TB), one of the worst globalkillers, with an estimated 1.7 million yearly deaths and a third of theworld’s population infected. The bacterium boasts one of nature’smost elaborate lipid metabolisms that is also key to its virulence,inherent drug resistance and ability to multiply within the macro-phage (20). There is a rapid development of multi drug-resistantstrains (MDR-TB) and extensively drug resistant TB strains (XDR-TB), resistant also to injectable second-line drugs, are emerging andpose a severe threat to TB control worldwide. Identification of newTB drugs and drug targets is imperative (21).

ResultsR2c-Like Proteins Form 2 Subgroups. When the R2c proteins werediscovered, there were only a handful of sequences available for thegroup (15). Now this number is above 50, allowing detailedsequence analysis. This reveals that R2c-like proteins actually form2 groups in a phylogenetic tree, 1 group including CtR2c and 1group including M. tuberculosis Rv0233 (Fig. 1). Alignments showthat the Rv0233 group does not contain the conserved C-terminaltyrosine, known to participate in radical transfer from R2 to thecatalytic R1 subunit and shown to be essential for activity in CtR2c(9, 22) (Fig. S1). Moreover, a number of organisms with fullysequenced genomes that contain proteins from the Rv0233 groupdo not contain any other protein component of a Class I RNRsystem. Together, this suggests that there may be functional differ-ences between the groups, despite an overall sequence similarityand conservation of active site and other key residues.

Author contributions: M.H. designed research; C.S.A. and M.H. performed research; C.S.A.and M.H. analyzed data; and M.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 3EE4).

1To whom correspondence should be addressed. E-mail: hogbom@dbb.su.se.

This article contains supporting information online at www.pnas.org/cgi/content/full/0812971106/DCSupplemental.

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Protein Production and RNR Assay. Rv0233 was produced as anN-terminal His6-affinity-tagged protein by overexpression in E. coli.M. tuberculosis H37Rv possesses 1 R1 homologue, nrdE, and 3genes that encode R2 protein homologues: nrdF1, nrdF2, andRv0233, presently annotated as nrdB. We were unable to obtain anyRNR activity above background from Rv0233 with M. tuberculosis R1(MtR1) either with or without the addition of 1 equivalent each ofFe(II) and Mn(II) per protein monomer while the MtR2, encoded bynrdF2, was highly active with MtR1 in the established [3H]CDP assay asalso shown previously (23). Activities obtained were (activity rel-ative to MtR1�MtR2 in %, average � 1 SD); MtR1�MtR2: 100 �7.1, MtR1�Rv0233: 0.55 � 0.12, MtR1�Rv0233�Fe(II)�Mn(II):0.50 � 0.083, MtR1 only, 0.43 � 0.083.

Overall Structure. The protein was crystallized and the structuresolved by SAD phasing to 1.9-Å resolution (Table 1). Rv0233 isa homodimer with the same interaction surface and geometry asR2s, producing the well-known heart-shaped dimer (Fig. 2A).The chain could be traced from residue 2 through 290, and theoverall structure is virtually identical to that of R2s. The core8-helix bundle is conserved, and the main differences areobserved in helices �D, �E, and the last 2 helices, �G and �H[nomenclature as defined in (24)] (Fig. 2B). The largest back-bone structural differences are observed in the N- and C-termini.The position of the C-terminus is interesting in comparison toR2s. The R2 C- terminus is known to interact with R1 and theC-terminal 30–40 residues are disordered in all R2 structuresdetermined to date. The last modeled residues, however, align inspace within a radius of only �5 Å, most likely relevant for theinteraction with R1. In Rv0233, the C-terminal 24 residues are alsodisordered but the last ordered residue is separated by �30 Åcompared with R2s. The difference in location of the C-terminusfurther supports the sequence and biochemical data showing thatthe protein is not an R2 component of a class I RNR.

Ligand Binding. A striking difference compared to R2 proteins is thepresence of a bound ligand that coordinates directly to the metal site(Fig. 3A). The ligand is modeled as myristic (C14) acid because ofthe fit to the electron density and the complete lack of H-bondinteractions between the ligand tail and the protein. The ligand isbound in a large continuous cavity, similar to the one observed in

BMMs with large substrates, for example, toluene monooxygenase(4, 25), extending from the metal site toward the protein surfacemade up of the loop linking helices �G and �H (Fig. 3B). The cavityis narrow and hydrophobic close to the metal site and widens, onceit has passed between helices �B and �E of the metal coordinating4-helix bundle, to produce a larger cavity with more H-bondpossibilities. The narrow part of the cavity is completely occupiedby the lipid ligand, while the wider part of the cavity is also solvatedby ordered water molecules. In the present structure, the cavity isclosed but conformational changes in the loop linking helices �Gand �H or rotamer changes of R59, E244, L248, or Y249 wouldopen the cavity to bulk solvent. The cavity is produced withoutsignificant movement of the protein backbone compared with R2s.This is achieved by numerous substitutions from larger to smallersidechains in combination with architectural differences in thesecond shell of cavity lining residues that allow a number of firstshell side chains to position differently, creating the cavity space.Based on the properties of the ligand binding cavity and the boundligand it seems most likely that the protein is involved in thebacterium’s lipid metabolism. Studies to define the in vivo substrateare initiated but complicated by the pathogenicity and very exten-sive lipid metabolism of M. tuberculosis, including several poorlydescribed mycobacterial-specific pathways.

Structure of the Heterodinuclear Metal Cofactor. Protein producedin standard rich LB media contains significant amounts of bothmanganese and iron, approximately 0.7 and 1.2 equivalents,respectively, as determined by ICP-SFMS. The Mn/Fe ratio isalso reflected in the relative intensity of the K-level X-rayemission lines from the crystal. Addition of 2 mM MnCl2 to theexpression medium shifts this relation close to unity. Thus, nomatter if the protein is produced at Mn:Fe ratios in the expres-

Fig. 1. Phylogenetic tree of R2 homologues where the canonical radicalharboring tyrosine is replaced by phenylalanine. The locations of C. tracho-matis R2c and M. tuberculosis Rv0233 are indicated.

Table 1. Crystallographic data and refinement statisticsfor Rv0233

Rv0233 � � 0.934 Å

Data collection MosflmBeamline ID14–1Wavelength, Å 0.934Space group P3221Cell parameters

Å 54.57; 54.57; 176.65° 90; 90; 120

Resolution, Å 40–1.90 (2.00–1.90)Completeness, % 99.9 (99.4)Redundancy 5.2 (4.4)Rsym, % 6.9 (36.2)I/�I 17.1 (3.5)Refinement statistics Refmac 5.4.0073Resolution, Å 40–1.9No. of unique reflections 24918No. of reflections in test set 1268Rwork, % 14.9Rfree, % 17.7No. of atoms

Protein 2319Metal ions 2Ligand 16Solvent 266

RMSD from ideal values*Bond length, Å 0.022Bond angle, ° 1.699

Ramachandran outliers, %† 1.5

*Ideal values from (37).†Calculated using a strict-boundary Ramachandran plot (38).

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sion media of roughly 1:10 (rich media) or 100:1 (rich media �2 mM MnCl2), it still contains close to 1 equivalent of both Mnand Fe per polypeptide.

The metal binding properties of the protein suggest to us that thisprotein, like the related C. trachomatis R2, contains the Mn/Fe-carboxylate redox cofactor. Anomalous diffraction difference mapsshow that the metal binding is specific with the manganese ionoccupying the site closest to the position of the radical harboringtyrosine in standard R2s, thus replacing Fe1 (Fig. 4A). The anom-alous data were collected on protein produced in Mn-supple-mented media. Still, there is no Mn anomalous signal from theFe-site above the noise level of the map. Similarly, there is no signof Fe binding in the Mn site. The metal binding is thus very specific,given that both metal ions are present in sufficient amounts. Themanganese ion has fewer carboxylate coordinations, despite thesymmetric arrangement of coordinating residues, this differencemay affect metal specificity. However, the coordination of theheterodinuclear cofactor is very similar to that observed in diironBMMs and R2s. In these systems there is also known to be a largeflexibility in metal coordination depending on oxidation state andcoordinating exogenous ligands (3, 4, 26, 27). The basis for thestrikingly strict metal specificity should lie in the metal free andreduced M(II)-M(II) forms because it is at this oxidation state thatthe metals bind to the protein. It seems very likely that the metal

specificity in this system involves outer-sphere effects. The presentstructure together with existing data on diiron systems shouldprovide a useful tool to study the fundamental processes of metalspecificity and redox tuning in proteins. The metal site surroundingsand H-bonding distances are depicted in Fig. 4B and C. Thenon-coordinating HxO species that is H-bonded to both the exog-enous ligand and Y175 refines to a distance of 3.0 Å from themanganese ion and does not seem to coordinate it directly;the electron density for this ligand is also somewhat weaker than forthe most well ordered water molecules, indicative of dynamics orpartial occupancy.

Tyrosine-Valine Crosslink in the Active Site. The catalytic potential ofthe metal site is manifested in the protein by the formation of an

Fig. 2. Structure of Rv0233. (A) Overall dimeric structure of the protein. (B)Superposition of Rv0233 (blue), E. coli R2 (green), and C. trachomatis R2c (red).Helices �D, �E, �G, and �H display the largest differences, the positions of theC-termini are indicated.

Fig. 3. Ligand binding and cavity in Rv0233. (A) Ligand binding and inter-action with the metal site shown by an Fo-Fc omit map for the ligandcontoured at 0.42 e�3. (B) The ligand-binding cavity in Rv0233 shown by theprotein molecular surface, the bound ligand is displayed as VdW spheres,ordered water molecules in the cavity are indicated. Residues R59, E244, L248,or Y249, restricting bulk solvent access are shown as sticks.

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unprecedented tyrosine-valine crosslink, likely generated duringone of the first redox cycles of the metal site. The phenolic oxygenof Y162 is covalently bound to C� of V71, connecting the metal sitecoordinating helices �B and �E (Fig. 5). The net chemical reactionis a 2-electron oxidation of the V71-Y162 side chain pair withremoval of 2 hydrogen atoms, resulting in the crosslink. With theexception of a few reported cases of peptides containing hydroxy-valine, this result is to our knowledge a previously undocumentedmodification of a valine side chain in a protein. Modification of thevery inert aliphatic side chain suggests that the Mn/Fe cofactor iscapable of similarly challenging 2-electron oxidations as BMMs.The use of the heterodinuclear site is thus not limited to generatingand storing a one-electron oxidizing equivalent as in CtR2c (16).Crosslinking of these amino acids, which are also conserved in thegroup, possibly prepares the active site for subsequent enzymaticchemistry (see Discussion).

We have considered if there could be alternative explanations forthe formation of the crosslink than via oxidation by the heterod-inuclear site. The only option in this case would be the action ofanother enzyme. The crosslink is deeply buried and, to be acces-sible, more than half of the protein would need to be unfolded.Moreover, this would also imply that the expression host, E. coli,possesses a system to form this crosslink in a M. tuberculosis proteinthat has no close homologues in E. coli. This possibility appearsextremely unlikely and cannot be considered a real option forcrosslink formation.

A Combination of Conserved Features from both R2s and BMMs. In thedirect vicinity of the metal site, the ligand-binding cavity is estab-lished by the substitution of a phenylalanine residue, which isabsolutely conserved in R2s, to an alanine (A171) conserved in theRv0233 group (Fig. S1). This positions the cavity in the same placeas the active site in BMMs. Moreover, in certain R2 mutants thatare engineered toward 2-electron chemistry, this phenylalaninebecomes hydroxylated (12, 28). In the present structure, the boundligand occupies the same position in space and is thus located at thepreferred location for substrate oxidation in diiron carboxylateproteins.

The hydrogen bonding network on the histidine side of the metalsite is known to control electron transfer and tuning between 1- and2-electron chemistry. In this area, the Rv0233 group displays a

Fig. 4. Rv0233 active site. (A) Anomalous difference maps. Purple: manga-nese anomalous difference density, contoured at 0.09 eÅ�3. Green: iron-specific ddano map, contoured at 0.07 eÅ�3. (B and C) Metal-site coordinationand hydrogen bonding network of conserved residues in the metal sitesurrounding, distances in Å.

Fig. 5. Covalent crosslink between V71 and Y162 shown by an Fo-Fc omitmap for the residues contoured at 0.42 e�3. The �-helical part of �E isillustrated by the n � 5 main chain H-bonds.

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composite structure of features otherwise unique to R2s or BMMs(Fig. S1 and Fig. 4 B and C). The residues preceding both metalcoordinating histidines are absolutely conserved as arginines inBMMs, indicating that they are essential for function or folding (4).The corresponding residues are mainly hydrophobic in R2s andR2cs. The Rv0233 group, like the BMMs has conserved positivelycharged residues in these positions, although the first is a lysine(K103). This indicates that these residues in BMMs and the Rv0233group are involved in electron transfer or redox tuning, rather thannecessary for folding because the structurally virtually identicalR2cs lack them. In addition, the Rv0233 group also possesses thetryptophan, W32 (W48 E. coli R2 numbering), normally a hallmarkof R2s and involved as a radical species in cofactor assembly (29).In BMMs, the large side chain of the arginine preceding the firstmetal coordinating histidine occupies the same position in space asthe conserved tryptophan in R2s. In Rv0233, on the other hand, thecorresponding K103 amine is involved in a �-cation interaction withthe pyrrole ring of W32, most likely tuning its chemical propertiesand redox potential. The hydrogen bonding network is thus alsovery similar to the one in the stearoyl-acyl carrier protein desatu-rases, another group of 2-electron, lipid-oxidizing, diiron carboxy-late proteins that display a different dimer interaction geometrythan R2s and BMMs (30). Y222 in CtR2c was recently shown tocontribute in mediating the 1-electron reduction of the Mn(IV)-Fe(IV) state to produce the active Mn(IV)-Fe(III) state. Mutationof this residue slows down the external 1-electron reduction, thusstabilizing the Mn(IV)-Fe(IV) intermediate (22). The correspond-ing residue is not conserved as an electron-relay competent residuein the Rv0233 group, something that may stabilize the Mn(IV)-Fe(IV) state and contribute to direct the protein to 2-electronoxidations.

DiscussionRecently, a group of R2 proteins was discovered (15). This groupuses a heterodinuclear Mn/Fe redox cofactor that, upon reactionwith molecular oxygen, yields a Mn(IV)-Fe(III) oxidation state thatis used in place of the diiron-tyrosyl radical system of standard R2s(16). This solution, which actually seems less complex, also appearsto be less sensitive to certain radical scavengers. Here, we show thatthe use of the Mn/Fe-carboxylate cofactor is not limited to the R2cproteins but is also present in a new group of ligand-bindingMn/Fe-carboxylate proteins. On the sequence level this group iseasily confused with the Mn/Fe-containing R2c proteins but thepresent structure allows assignment of available sequences to thedifferent groups. There are a number of sequence particulars thatstrongly indicate that the proteins in the new group are all ligand-binding oxidases, and we thus propose that they are denoted‘‘R2-like ligand binding oxidases.’’ The in vivo substrates andproducts are presently unknown, and may well differ within thegroup. It seems most likely that the proteins are hydrocarbonoxidases, possibly involving oxygen insertion. The potential forchallenging 2-electron oxidations by the heterodinuclear cofactor isdemonstrated by the formation of an unprecedented tyrosine-valine crosslink in the active site. This shows that the Mn/Fecofactor has a richer chemical repertoire than the generation andstorage of a one-electron oxidizing equivalent, as in CtR2c, and maybe similarly versatile as the diiron-carboxylate cofactor.

We also describe the detailed structure of this cofactor. Eventhough this structure is for a protein that is not an R2 the extensivestructural similarities between the groups strongly suggest that theR2c proteins also have the same arrangement with the Mn ionsubstituting for Fe1. Since it is known that the manganese assumesan (IV) oxidation state and serves as the radical initiator in theactive state of CtR2c its position has great importance for theradical transfer in these systems and should also have implicationsfor the details of radical transfer in the canonical diiron R2 proteins.

Because the residues involved in the tyrosine-valine crosslink areconserved, it likely has relevance for protein function, this role is not

obviously apparent. Some interesting features in relation to BMMscan however be noted. Binding of the regulatory protein in BMMsinduces structural changes in the active site that increase oxygenreactivity and turnover as well as influence the regiospecificity forhydroxylation (3). CD and MCD studies on MMO show that thisis mainly a result of structural changes around Fe2, in particular ofE209, corresponding to E167 in Rv0233 (31). In the complexbetween phenol hydroxylase and its regulatory protein the �E helixadopts a �-helical structure in this region. It was proposed that thisfeature might mediate the effect of the regulatory protein to theactive site (25). A recent study of toluene-4-monooxygenase showsthat effector protein binding induces a number of structural changesin the metal site coordinating helices, especially �E, leading tochanges in both metal ligation and the active-site channel (32). TheY-V crosslink in Rv0233 puts a strict geometric restraint betweenhelices �B and �E and establishes a �-helix in �E, comprising 2 fullturns and including E167, illustrated by the n � 5 main chainH-bonds in Fig. 5. The �E helices in BMMs are more distorted interms of straightness than �E in Rv0233; still it is noteworthy thatthe same segment adopts a �-helical structure. A possible conse-quence is that the crosslink functions as a poor man’s regulatoryprotein, imposing geometric restraints that fix the helix in its�-conformation and thus lock the protein in one of several statesotherwise controlled by the regulatory protein in BMMs. It remainsto be shown whether this adduct has additional functions or takespart in the chemistry as a cofactor.

Reconstitution of the Mn/Fe cofactor in CtR2c involves aMn(IV)-Fe(IV) oxidation state (33). This is interesting because theFe(IV)-Fe(IV) state has never been observed in an R2 proteinwhile methane monooxygenase is known to use this intermediatefor substrate oxidation. The phenolic oxygen of the cross-linkedY162 is located 5.1 Å from the iron ion. This is the same distanceas the buried radical harboring tyrosine in canonical R2s is to Fe1(ranging from � 4.6–6.6 Å depending on species and oxidationstate). By analogy with these systems we hypothesize that thecovalent link may be created via a mechanism involving a Y162radical produced by the Mn(IV)-Fe(IV) oxidation state (Fig. S2).

Interestingly, the phenolic oxygen of another conserved tyrosine,Y175, present in all proteins in the Rv0233 group, but conserved asPhe in R2cs and standard R2s, is H-bonded to the metal site ligandE202 and via a water molecule to E68 (Fig. 4B and C). The phenolicoxygen of Y175 lines the substrate-binding cavity and is positioned4.9 Å from the manganese ion. Similarly to the radical harboringtyrosine in R2s, Y175 is also linked by H-bonds to the metal site.It thus seems reasonable that Y175 can become oxidized to a radicalspecies by the metal site. However, unlike the R2s that bury thetyrosyl radical in a hydrophobic pocket in the protein, this residueis exposed to the substrate. In the present structure Y175 is alsoH-bonded to the exogenous ligand via a water molecule. This opensthe possibility that substrate oxidation and formation of the cova-lent crosslink proceed via similar mechanisms involving tyrosylradical-linked high valent metal site intermediates. Based on thepositions of the HxO species, reactive metal-oxygen intermediatesproduced by dioxygen cleavage are expected to reside on thesubstrate-binding side of the metal site close to Y175, providingpossibilities for oxygen insertion into the substrate.

From the pattern of sequence conservation it seems that theprotein is a hybrid between BMMs and R2s. The conserved featuresof the 2 diiron systems that are merged in Rv0233 must, however,be interpreted in light of that the protein has a high specificity forMn and Fe and that it is the heterodinuclear cofactor that producesthe tyrosine-valine crosslink. The R2-like ligand binding oxidasesdescribed here close the circle of a group of proteins that performboth fascinating and important chemistry. They merge structuraland functional features from 2 well-studied families, the R2s andBMMs. This group of proteins should provide a key tool toconsolidate and test theories about mechanistic differences andsimilarities.

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Evolution of BMMs is believed to have ensued via a geneduplication of a diiron carboxylate protein and subsequent diver-gence into the catalytic �-subunit and the non-catalytic �-subunit,while retaining the overall fold and dimer interaction geometry.Accessory protein subunits were likely acquired via horizontal genetransfer, which also largely characterizes the spread of BMMs (6,11). The Mn/Fe-carboxylate proteins could represent structural andfunctional homologues of an ancient ancestor of R2s and BMMsand Mn/Fe could be considered a possible candidate for theancestral cofactor. Although the evolutionary relationship is clearlythe topic for more detailed analysis, it is apparent that bothfunctions can be housed in very similar homodimeric proteinscaffolds with a heterodinuclear Mn/Fe-carboxylate cofactor.

Materials and MethodsDetailed materials and methods are described in SI Materials and Methods.

Bioinformatics. Sequences encoding R2 homologues but lacking the canonicalradical harboring tyrosine were collected by sequence database searching.

Cloning, Protein Expression, Purification, Enzymatic Assays, and Metal Analysis.The Rv0233 gene was PCR cloned from M. tuberculosis strain H37Rv (20) andoverexpressed in E. coli BL21(DE3) grown in LB medium either without metalsupplement or with the addition of 2 mM MnCl2. Protein was purified byaffinity and size exclusion chromatography. Ribonucleotide reductase enzy-matic activity for M. tuberculosis R1 with Rv0233 was measured using theestablished [3H]CDP assay. Initial indication that the protein contained more

than one metal was obtained by a simple combined luminescence and color-imetric assay (34). Quantitative metal analysis was performed using induc-tively coupled plasma sector field mass spectrometry. The intensity of theX-ray K-level emission lines were also used to estimate the relative amount ofMn and Fe in the crystal as well as to verify that the crystallization process didnot impose any significant enrichment of protein containing a particularmetal.

Crystallization, Data Collection, and Structure Determination. Rv0233 wascrystallized using the vapor diffusion method. Diffraction data were collectedat the ESRF synchrotron in Grenoble, France. Data collection statistics areshown in Tables 1, S1, and S2. The intrinsic metal cofactor of the protein wasused to phase the data by means of single-wavelength anomalous dispersionmethods using anomalous data collected at the high energy side of the ironedge. Model statistics are presented in Table 1. To determine metal identity inthe different binding sites, anomalous diffraction data were collected at thehigh-energy side of the Mn-edge (� � 1.8 Å) and the high-energy side of theFe-edge (� � 1.7 Å) (Table S2). Anomalous difference model phased Fourier(DANO) maps were calculated with FFT (35). At � � 1.8 Å, manganese, but notiron, displays an anomalous signal and these data were used to determine theposition of the Mn ion. Since both manganese and iron display anomaloussignals at � � 1.7 Å, an iron-specific ‘‘difference DANO’’ map was calculatedusing both datasets according to (36). Coordinates and structure factors aredeposited in the PDB with id 3EE4.

ACKNOWLEDGMENTS. We are very grateful to T. Alwyn Jones for support anddiscussions. This work was supported by grants from the Swedish ResearchCouncil and the Swedish Foundation for Strategic Research to M.H. and T.A.J.and the Knut and Alice Wallenberg Foundation to M.H.

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