ribonucleotide reductases 
 ( rnrs ) perform an essential function 
 in all organisms , catalyzing the conversion of ribonucleotides into 
 deoxynucleotides and providing the monomeric precursors required for 
 dna synthesis and repair ( chart 1 ) .
all rnrs initiate nucleotide reduction via a transient protein - based 
 thiyl radical that abstracts a hydrogen 
 atom from the 3-position of the nucleotide .
rnrs have been divided into three classes ( i , ii , and iii ) 
 based on the metallo - cofactors required to generate this initiating 
 thiyl radical .
this paper focuses on the e. coli class iii rnr and the identification of a 
 new radical species involving a three - electron bond between a cysteine 
 and a methionine residue playing a role in the reductive half - reaction . 
 despite the sequence differences between the three rnr classes , the 
 first evidence for the involvement of thiyl radicals in the reductive 
 half - reaction of all classes
two of them are class i rnrs , 
 one with a diferric - tyrosyl radical ( y ) cofactor ( class ia ) 
 and one with a dimanganese - y cofactor ( class ib ) .
the 
 ia enzyme is the workhorse in dna replication , while the ib rnr is 
 expressed under iron limitation and oxidative stress .
the third rnr , 
 which is the focus of this paper , is a class iii enzyme and is expressed 
 only under anaerobic conditions .
nucleotide reduction is catalyzed 
 by the 80 kda nrdd , which houses the o2-sensitive glycyl 
 radical ( g ) that is generated 
 by the 17.5 kda activating enzyme nrdg via chemistry involving s - adenosylmethionine ( sam ) and a [ 4fe4s ] cluster 
 ( chart 1 ) .
the g , as with 
 the dimetallo - ys in the class ia and ib rnrs and adenosylcobalamin 
 in the class ii rnr , is reversibly involved in generation of the thiyl 
 radical ( chart 1).chart 1the class i , ii , and iii rnrs catalyze 
 the conversion 
 of nucleoside di or triphosphates to deoxynucleotides .
they differ 
 in the metallo - cofactor that initiates the reduction process and in 
 the reductant itself . reproduced from rhodes et al .
the class i , ii , and iii rnrs catalyze 
 the conversion 
 of nucleoside di or triphosphates to deoxynucleotides .
they differ 
 in the metallo - cofactor that initiates the reduction process and in 
 the reductant itself . reproduced from rhodes et al .
the class i , ii , and iii rnrs catalyze 
 the conversion 
 of nucleoside di or triphosphates to deoxynucleotides .
they differ 
 in the metallo - cofactor that initiates the reduction process and in 
 the reductant itself . reproduced from rhodes et al .
recent studies on the class 
 ia rnr and earlier studies on the class 
 ii enzyme established that the chemistry 
 of nucleotide reduction is fast ( 100 
 s ) , and in both cases it is masked in the steady 
 state by conformational gating ( kcat of 5 s ) . the mechanism 
 of nucleotide reduction by the class i and ii rnrs has been investigated 
 using many methods resulting in the model shown in figure 1a , b .
the overall reaction 
 is divided into two half reactions . in the first half - reaction ( figure 1a ) , a 3-nucleotide radical ( 2 )
is generated 
 by 3-hydrogen atom abstraction by the transient thiyl radical 
 located on the top face of the sugar .
water loss is then facilitated 
 by protonation by a bottom face cysteine , in an irreversible step , 
 to generate intermediate 3 .
these first two steps are 
 likely facilitated by removal of the proton of the 3-oh group . in the second half - reaction ( figure 1b ) , intermediate 3 is reduced to the 
 3-keto - deoxynucleotide ( 4 ) concomitant with formation 
 of the disulfide anion radical which 
 then , in a proton - coupled electron - transfer step , generates the 3-deoxynucleotide radical 5 . in the final step , the hydrogen atom that was initially removed 
 from the 3-position of the nucleotide
mechanistic model for nucleotide reduction by 
 the e. coli class ia rnr and e. coli ( or bacteriophage t4 ) class iii rnr .
( a ) 
 first half - reaction common 
 to all rnrs ; ( b ) second half - reaction of class ia rnr ; and ( c ) second 
 half - reaction of class iii rnr .
the mechanism of nucleotide reduction by the class iii rnrs 
 has 
 been less well characterized , but studies support a similar sequence 
 of events for the first half - reaction ( figure 1a ) .
a distinction between the rnr classes is 
 apparent in the proposed mechanism of the second half - reaction ( compare 
 figure 1b , c ) , as a pair of conserved , bottom 
 face , cysteine residues are essential for nucleotide reduction by 
 the class i and ii rnrs , while formate 
 is the reductant in the e. coli class 
 iii rnr .
studies on the class iii rnr 
 in d2o demonstrated that solvent replaces the 2-oh 
 with retention of configuration and that a small amount of exchange 
 of deuterium into the 3-position of the product takes place .
these results are similar to previous studies 
 with the class i and ii rnrs . studies with [ h]-formate also provided insight about 
 the reduction process .
first , h was found in the solvent , consistent with the oxidation of formate 
 to a co2 via the conserved bottom 
 face cysteine .
second , an unusual [ h]-(kcat / km ) isotope effect of 
 2.3 was observed , requiring exchange of formate between the active 
 site and solvent prior to cleavage of the formate c  h bond 
 and after the first irreversible step of nucleotide reduction , thought 
 to be h2o loss .
thus , formate appears to be able to enter 
 and leave the active site in the middle of nucleotide turnover .
a comparison of the active site structures of the e. coli class ia 2 ( nrda ) and the 
 bacteriophage t4 class iii 2 ( nrdd , figure s1a ) reveals similar 10-stranded / 
 barrels containing a finger loop with the essential cysteine c290 
 ( equivalent to c439 in e. coli nrda , 
 c384 in e. coli nrdd ) at its tip , that 
 generates the substrate radical . of the 
 two cysteines that donate the reducing equivalents in the class
i 
 and ii rnrs ( c225 and c462 in e. coli nrda ) , only c79 ( c225 in e. coli nrda , 
 c175 in e. coli nrdd ) is conserved 
 in the class iii enzyme .
mutagenesis studies showed that both c79 
 and c290 are essential for catalysis in the class iii enzyme .
additional similarities between the class 
 i , ii , and iii rnrs have been 
 identified using mechanism - based 
 inhibitors , 2-chloro and fluoro 2-deoxynucleotides , 
 that can also function as alternative substrates ( figure 2 ) .
all rnrs react with these analogues to form 13 , which can partition between the normal reduction reaction 
 to generate the deoxynucleotide product ( figure 1b ) , or reduction from the top face or the bottom face of the sugar 
 to generate a 3-keto - deoxynucleotide ( 14 ) , which 
 dissociates from the active site and decomposes nonenzymatically to 
 produce the nucleobase and pyrophosphate ( tripolyphosphate ) . in the 
 case of the class i and ii rnrs , the furanone ( 15 ) has 
 also been identified , and its reaction with the enzyme has been shown 
 to result in their time - dependent inactivation .
similarities in the reactivity of all three classes of 
 rnrs toward these substrate analogues suggest a common first half - reaction 
 ( figure 1a ) and provide a mechanism by which 
 nucleobase ( n ) can be formed ( figure 2 ) .
mechanistic 
 model for nucleotide reduction by the e. coli class ia rnr with a mechanism - based inhibitor 
 or an active site mutant ( zh ) with cdp . reported here
are our studies of the reaction of e. coli class iii rnr that have provided insight 
 about the reductive half - reaction of this and the class i and ii rnrs . 

incubation of the class iii rnr with ctp ( substrate ) and atp ( allosteric 
 effector ) in the absence of formate leads to the disappearance of 
 the g , concomitant with formation of a new radical and release 
 of cytosine ( cyt ) , a known breakdown product of 3-keto - deoxycytidine 
 ( figure 1c , 7 , and figure 2 , 14 ) .
subsequent addition of formate leads to the disappearance of the 
 new radical , recovery of g , and formation of dctp and a small 
 amount of cyt .
isotopic labeling of nrdd with [ -h]-cysteine , [ -h]-methionine and [ ,-h]-methionine , in combination with epr spectroscopy of the 
 new radical , identifies this species as a thiosulfuranyl radical ( figure 1c , 8) , generated from the c175 thiyl 
 radical stabilized by an interaction with the conserved m382 .
the 
 role of the thiosulfuranyl radical in the class iii rnr second half - reaction 
 ( figure 1c ) and its relationship to the disulfide 
 radical anion in the second half - reaction of the class ia rnr ( figure 1b ) is discussed .
all chemical reagents 
 were purchased from sigma  aldrich , unless otherwise indicated . 

uv  vis 
 absorption spectroscopy was performed on an agilent 8453 diode array 
 spectrophotometer .
anaerobic procedures were carried out in a custom - designed 
 mbraun glovebox in a cold room at 4 c or at room temperature 
 ( rt ) .
all solutions and proteins were made anaerobic on a schlenk 
 line by 3 cycles of evacuation ( 5 min ) followed by flushing with ar 
 gas ( 10 min ) before being brought into the glovebox .
the plasmids 
 prss and pn9 containing the genes for e. coli nrdd and nrdg , respectively , were gifts 
 from professor marc fontecave , collge de france ( paris , france ) . the synthesis was carried out by minor modifications 
 of the procedure of lohman et al .
a reaction 
 mixture ( 10 ml ) containing 5-[h]-cdp ( 2 mm , vitrax , specific 
 activity of 26880/nmol ) , pep ( 4 mm ) , tris - hcl ( 50 mm , ph 7.5 ) , kcl 
 ( 80 mm ) , mgcl2 ( 20 mm ) , and pyruvate kinase ( rabbit muscle , 
 120 u / ml ) was incubated at 37 c for 1 h. the resulting 5-[h]-ctp was purified on a deae column ( 60 ml , 10  2 cm ) 
 washed with water ( 50 ml ) , and eluted with a 400  400 ml linear 
 gradient from 0 to 750 mm triethylammonium bicarbonate ( teab ) .
the 
 triphosphate containing fractions eluting at 550 mm teab were combined , 
 and the solvent removed in vacuo .
the product 5-[h]-ctp was isolated in 87% yield and the structure confirmed 
 by h- and p nmr .
the 5-[h]-ctp 
 used in all experiments had a specific activity of 2860 cpm / nmol .
the plasmid prss contains a 
 1800 bp insert between the promoter and nrdd start 
 codon .
an ndei restriction site was introduced at the 5 position 
 of the nrdd orf by site - directed mutagenesis using 
 the primer gttcttaaaaatatggagcgcatatgacaccgc 
 ( mutation underlined ) .
pcr was carried out using pfuultraii polymerase 
 ( stratagene ) according to the manufacturer s protocol , followed 
 by dpni digestion of the methylated template plasmid .
the resulting 
 plasmid was digested with ndei and ecori ( neb ) and 
 the nrdd fragment was ligated into pet24a ( novagen ) , 
 which was linearized with the same restriction enzymes , to give pet24a - nrdd .
the nrdd sequence and all cloned 
 or mutant sequences were confirmed by dna sequencing at the massachusetts 
 institute of technology biopolymers laboratory . to facilitate insertion of nrdd into ptrchisa ( invitrogen ) without an n - terminal his6-tag ,
an ndei restriction site was introduced into the plasmid by 
 site - directed mutagenesis using the primer cgattaaataaggaggaataacatatgtatcgattaaataagg ( mutation underlined ) , and an 
 internal ndei restriction site present in the plasmid was mutated 
 using the primer ggtatttcacaccgcacatggtgcactc . 

the nrdd fragment 
 was excised from pet24a - nrdd using the same restriction 
 enzymes and ligated into ptrc to give ptrc - nrdd .
the s386e mutation was introduced 
 by site - directed mutagenesis of the plasmids pet24a - nrdd and ptrc - nrdd using the primer ccgatgggctgccgcgagttcctcggcgtgtggg ( mutation underlined ) .
pet24a - nrdd was transformed 
 into bl21 ( de3 ) cells ( invitrogen ) , grown on lb - agar 
 plates with 50 g / ml kanamycin ( kan ) at 37 c .
a single 
 colony was inoculated into 5 ml of lb ( 50 g / ml kan in all growths ) , 
 grown at 37 c until saturated ( 12 h ) , and transferred into 1.5 
 l of lb supplemented with 50 m zinc acetate in a 6 l erlenmeyer 
 flask .
the culture was grown at 37 c with shaking at 220 rpm . 
 at od600  0.6 ,
the temperature was lowered to 25 
 c and isopropyl -d-1-thiogalactopyranoside ( iptg , 
 promega ) was added to a final concentration of 1 mm .
after 14 h , the 
 cells were pelleted by centrifugation ( 5000  g , 10 min , 4 c ) and frozen at 80 c .
cell paste 
 ( 7.1 g ) was resuspended in 35 ml of buffer a ( 50 mm tris , 
 5% glycerol ( bdh chemicals ) , 5 mm dithiothreitol ( dtt , promega ) , ph 
 7.5 ) containing 1 mm pmsf .
the cells were lysed by a single passage 
 through a french pressure cell ( 14  000 psi ) .
dna was precipitated 
 by dropwise addition of 0.2 vol of buffer a containing 6% ( w / v ) streptomycin 
 sulfate .
the mixture was stirred for an additional 10 min , and the 
 precipitated dna was removed by centrifugation ( 20  000  g , 10 min , 4 c ) .
the solution was 
 stirred for an additional 20 min , and the precipitated protein was 
 isolated by centrifugation ( 20  000  g , 10 min , 4 c ) .
the pellet was dissolved in 20 ml of 
 buffer a containing 0.8 m 
 ( nh4)2so4 and loaded onto a butyl - sepharose 
 column ( 2  16 cm , 50 ml ) .
the column was eluted with 2 column 
 volumes ( cv ) each of buffer a containing 0.8 , 0.6 , 0.4 , 0.2 , and 0.0 
 m ( nh4)2so4 , and the fractions were 
 analyzed by sds - polyacrylamide gel electrophoresis ( sds - page , 10% 
 gel ) .
this analysis led to rechromatography of the protein on the 
 butyl - sepharose column with a gentler step - gradient .
the fractions 
 eluting at 0.60.4 m ( nh4)2so4 were pooled and precipitated with ( nh4)2so4 ( 60% saturation ) .
the pellet was redissolved in buffer a 
 containing 0.8 m ( nh4)2so4 and loaded 
 onto the butyl - sepharose column ( 2  16 cm , 50 ml ) .
the column 
 was then eluted with 2 cv each of buffer a containing 0.8 , 0.7 , 0.6 , 
 0.5 , and 0.4 m ( nh4)2so4 . the fractions 
 eluting at 0.70.5 m ( nh4)2so4 were pooled and precipitated with ( nh4)2so4 ( 60% saturation ) .
the pellet was dissolved in 100 ml 
 of buffer a and was loaded onto 
 a deae - sepharose column ( 2  16 cm , 50 ml ) and washed with 100 
 ml of buffer a. the flow - through and wash fractions were pooled and 
 precipitated with ( nh4)2so4 ( 60% 
 saturation ) . the pellet was dissolved in a minimal volume of buffer 
 a ( 10 ml ) and desalted using a sephadex g-25 column ( 2  50 cm , 
 150 ml ) .
the protein - containing fractions were pooled and concentrated 
 by ultrafiltration ( amicon ym-30 , millipore ) .
this procedure yielded 
 55 mg nrdd per g of cells ( 280 = 77  020 mcm ) , judged pure by sds - page .
pn9 was transformed 
 into bl21 ( de3 ) cells ( invitrogen ) , grown on lb - agar plates with 100 
 g / ml ampicillin ( amp ) at 37 c .
a single colony was inoculated 
 into 5 ml of lb ( 100 g / ml amp in all growths ) , grown at 37 
 c until saturated ( 12 h ) , and transferred into 6  1.5 
 l of lb in 6 l erlenmeyer flasks .
the cultures were grown at 37 c 
 with shaking at 220 rpm . at od600  0.7 ,
the temperature 
 was lowered to 28 c , and iptg was added to a final concentration 
 of 1 mm .
after 12 h , the cells were pelleted by centrifugation ( 4000 
  g , 10 min , 4 c ) and frozen at 80 
 c .
cell paste ( 12.9 g ) was resuspended in 50 ml of buffer 
 b ( 30 mm tris , 50 mm kcl , 10 mm dtt , ph 7.5 ) containing 0.1 mm pmsf . 

the cells were lysed by a single passage through a french pressure 
 cell ( 14  000 psi ) .
dna was precipitated by dropwise addition 
 of 0.2 vol of buffer a containing 6% ( w / v ) streptomycin sulfate .
the 
 mixture was stirred for an additional 10 min , and the precipitated 
 dna was removed by centrifugation ( 15  000  g , 10 min , 4 c ) .
the solution was stirred for an 
 additional 20 min , and the precipitated protein was isolated by centrifugation 
 ( 15  000  g , 40 min , 4 c ) .
the pellet was dissolved in 10 ml of buffer b and desalted using 
 a sephadex g-25 column ( 5  50 cm , 1 l ) equilibrated with buffer 
 b. the colored fractions were combined and loaded onto a deae - sepharose 
 column ( 5  10 cm , 200 ml ) and washed with 300 ml of buffer b. 
 the flow - through and wash fractions were pooled and precipitated with 
 ( nh4)2so4 ( 40% saturation ) .
the pellet 
 was dissolved in 4 ml of buffer b and loaded onto a sephadex g-75 
 column ( 3  100 cm , 700 ml ) equilibrated with buffer b. fractions 
 ( 15 ml ) were collected and analyzed by sds - page .
the fractions containing 
 nrdg were pooled and concentrated by ultrafiltration ( amicon ym-10 ) , 
 yielding 4 mg nrdg per g of cells ( 280 = 28480 mcm ) , pure by sds - page .
the procedure 
 was carried out in a glovebox in a 4 c cold room .
solutions 
 of na2s and of fe(nh4)2(so4)2 in water ( 100 mm ) were freshly prepared in the glovebox . 
 a solution of nrdg ( 250 m , 1 ml ) was made anaerobic on a schlenk 
 line and brought into the glovebox .
a solution of dtt ( 100 mm ) was 
 added to 10 mm , followed by ordered addition of a solution of na2s ( 5 equiv ) and fe(nh4)2(so4)2 ( 5 equiv ) , and incubation for 12 h. the solution was 
 concentrated to 200 l by ultrafiltration ( amicon ym-30 ) .
edta 
 ( 5 equiv ) was then added , and the solution was desalted using a sephadex 
 g-25 column ( 1  9 cm , 7 ml ) equilibrated with triethanolamine 
 ( tea ) buffer ( 30 mm , ph 7.5 ) .
the final material typically contained 
 2 atoms of fe per peptide determined by the ferrozine assay . in order to carry out reactions in the absence of 
 formate
, substitutes 
 were used for components of the nrdd storage buffer found to contain 
 a formate contaminant ( tris base , glycerol , and dtt , see results section ) .
inositol and tcep , which contain 
 undetectable levels of formate , effectively replaced glycerol and 
 dtt in preserving nrdd activity .
tris buffer was replaced with bicine , 
 which also served as a coreductant for the photoreduction system . 

bicine was later found to contain small amounts of formate and was 
 thus replaced with tea for experiments where complete removal of formate 
 is desired . to generate nrdd - g ,
a solution of nrdd ( 200 
 m , 1.5 ml ) was concentrated to 300 l by ultrafiltration 
 ( amicon ym-30 ) .
the protein was exchanged into bicine buffer ( 30 mm , 
 ph 7.5 ) , 3% inositol , 5 mm tris(2-carboxyethyl)phosphine ( tcep ) , using 
 a sephadex g-25 column ( 1  20 cm , 15 ml ) .
the protein - containing 
 fractions were pooled and made anaerobic on a schlenk line and brought 
 into a glovebox in a 4 c cold room .
a 50 l mixture 
 of nrdd ( 100 m ) , nrdg ( 10 m ) , sam ( 1 mm ) , bicine buffer 
 ( 30 mm , ph 7.5 ) and rose bengal ( 50 m ) was exposed to a fluorescent 
 lamp in the glovebox for 2 h. for inspection by x - band epr spectroscopy , 
 the solution was diluted to 250 l with bicine buffer ( 30 mm , 
 ph 7.5 ) , 3% inositol to give a final concentration of 20 m 
 nrdd , and sealed in an epr tube with a rubber stopper .
the solution 
 was quenched in liquid n2 immediately after removal from 
 the glovebox .
the amount of g in the solution was determined 
 by comparing the epr signal intensity to that of a cuso4 standard . a typical yield of 0.450.50 
 radicals per nrdd polypeptide
the assay mixture 
 in 100 l contained nrdd ( 0.2 m ) , atp ( 1 mm ) , 5-[h]-ctp ( 1 mm , 2860 cpm / nmol ) in tris ( 30 mm , ph 7.5 ) , kcl 
 ( 30 mm ) , mgso4 ( 10 mm ) , and hco2na ( 10 mm ) and 
 was incubated at 25 c .
four , 20 l aliquots were removed 
 at 1 min intervals and quenched with 2% perchloric acid ( 20 l ) . 

subsequent to removal of the phosphates using calf intestine alkaline 
 phosphatase ( roche ) , dctp formation was analyzed by the method of 
 steeper and steuart .
the specific activity of nrdd 
 is 1500 u / mg , consistent with literature values .
the method 
 of steeper and steuart 
 involves chromatography on a dowex-1-borate column where the cytidine 
 is retained as a complex with borate , while dc is eluted . to assay 
 for cyt released from 3-keto - dctp , the dephosphrylation step 
 was omitted .
controls showed that ctp and dctp are retained on the 
 dowex anion - exchange column , while the cyt passes directly through 
 the column .
5-[h]-ctp was lyophilized before the experiments , 
 eliminating any h2o that would also pass directly 
 through the column .
the final reaction mixture at 
 4 c contained in a volume of 
 250 l : nrdd ( 40 m , activated as described above ) , ctp 
 ( 1.5 mm ) , atp ( 1.5 mm ) , bicine ( 30 mm , ph 7.5 ) , kcl ( 30 mm ) , mgso4 ( 10 mm ) , and 3% inositol .
the reaction was initiated by addition 
 of nrdd , and the sample was rapidly transferred and sealed in an epr 
 tube with a rubber stopper , removed from the glovebox , and frozen 
 in liquid n2 .
the total reaction time was 1 min for nrdd - wild - type 
 ( wt ) and 60 min for nrdd-(s386e ) .
for reaction with formate , 
 nrdd was reacted first with ctp and atp for 1 min at 4 c as 
 described above , followed by addition of sodium formate to 10 mm ( 2.5 
 l , 1 m ) .
continuous wave ( cw ) x - band 
 epr spectra were recorded at 77 k in the mit department of chemistry 
 instrumentation facility on a bruker esp-300 x - band spectrometer equipped 
 with a quartz finger dewar filled with liquid n2 .
experimental 
 conditions were as follows : microwave frequency , 9.45 ghz ; modulation 
 amplitude , 0.15 mt ; modulation frequency , 100 khz ; time constant , 
 5.12 ms ; and scan time , 41.9 s. a microwave power of 10 w and 
 an average of 10 scans was used for g , while a power of 160 
 w and 100 scans was used for the thiosulfurnanyl radical .
a solution of nrdd and nrdg was concentrated by ultrafiltration ( amicon 
 ym-30 ) in a glovebox in a 4 c cold room .
the final mixture for 
 g generation ( 5 l ) contained nrdd ( 800 m ) , nrdg 
 ( 80 m ) , sam ( 1.5 mm ) , bicine ( ph 7.5 , 30 mm ) , kcl ( 30 mm ) , 
 mgso4 ( 10 mm ) , rose bengal ( 50 m ) , and 3% inositol . 

the mixture was exposed to the fluorescent lighting in the glovebox 
 at 4 c for 2 h to generate g. the reaction with the 
 nucleotide was initiated by adding a solution ( 1 l ) containing 
 ctp and atp in reaction buffer , to give a final reaction concentration 
 of 2 mm each .
loading and freezing the small 140 ghz epr tubes ( i d 
 0.5 mm , effective sample volume 200 nl ) could not be done as swiftly 
 as with the x - band tubes , and the total reaction time was extended 
 to 2.5 min at 4 c .
echo - detected 
 140 ghz epr 
 spectra were obtained on a spectrometer developed by smith et al .
pulsed epr spectra were acquired at a temperature 
 of 85 k using a hahn echo pulse sequence ( /2 pulse = 35 ns 
 and  = 250 ns ) .
the h resonance frequency of a small water sample 
 that resides just below the sample space in the magnet was used to 
 set the magnetic field .
ptrc - nrdd or ptrc - nrdd-(s386e ) was transformed into the e. coli cysteine auxotroph jw3582 - 2 ( yale e. coli genetic stock center ) , containing the mutation cyse720::kan .
a single colony was inoculated into 5 ml of 
 lb ( 100 g / ml amp in all growths ) , grown at 37 c until 
 saturated ( 12 h ) , harvested by centrifugation ( 3000  g , 10 min , 4 c ) , and transferred into m9 medium supplemented 
 with l - amino acids and cofactors .
the growth medium contained 
 in 1 l : na2hpo4 ( 6 g ) , kh2po4 ( 3 g ) , nh4cl ( 1 g ) , nacl ( 1 g ) , glycerol ( 4 g ) , 
 mgcl2.6h2o ( 210 mg ) , and cacl2.2h2o ( 14 mg ) , alanine ( 0.50 g ) , arginine ( 0.40 g ) , aspartic acid 
 ( 0.40 g ) , asparagine ( 0.40 g ) , glutamine ( 0.4 g ) , sodium glutamate 
 ( 0.74 g ) , glycine ( 0.55 g ) , histidine hydrochloride ( 0.13 g ) , isoleucine 
 ( 0.24 g ) , leucine ( 0.23 g ) , lysine hydrochloride ( 0.43 g ) , methionine 
 ( 0.25 g ) , phenylalanine ( 0.15 g ) , proline ( 0.10 g ) , serine ( 2.10 g ) , 
 threonine ( 0.23 g ) , tryptophan ( 0.06 g ) , valine ( 0.23 g ) , thiamine 
 ( 50 mg ) , and racemic [ ,-h]-cystine 
 ( 120 mg , 98% , cambridge isotope laboratories ) .
the cultures were grown 
 at 37 c with shaking at 220 rpm . at od600  
 0.6 ,
the temperature was lowered to 25 c , and iptg was added 
 to a final concentration of 1 mm .
after 14 h , the cells were pelleted 
 by centrifugation ( 5000  g , 10 min , 4 c ) 
 and frozen at 80 c , yielding 2 g of cell paste 
 per l of culture .
pet24a - nrdd or pet24a - nrdd-(s386e ) was transformed into bl21 
 ( de3 ) cells , grown on lb - agar plates with 50 g / ml kan .
a single 
 colony was inoculated into 5 ml of lb ( 50 g / ml kan in all growths ) , 
 grown at 37 c until saturated ( 12 h ) , harvested by centrifugation 
 ( 3000  g , 10 min , 4 c ) , and transferred 
 into m9 medium containing in 1 l : na2hpo4 ( 6 
 g ) , kh2po4 ( 3 g ) , nh4cl ( 1 g ) , nacl 
 ( 1 g ) , glycerol ( 4 g ) , mgcl2.6h2o ( 210 mg ) , 
 cacl22h2o ( 14 mg ) , and thiamine ( 50 mg ) . 

the cultures were grown at 37 c with shaking at 220 rpm . at 
 od600 0.3 ,
the l - amino acids lysine ( 0.1 
 g ) , phenylalanine ( 0.1 g ) , threonine ( 0.1 g ) , isoleucine ( 0.05 g ) , 
 leucine ( 0.05 g ) , and valine ( 0.05 g ) were added .
additionally , 50 
 mg of [ -h]-methionine ( 98% , cambridge isotope laboratories ) 
 or [ ,-h]-methionine ( 98% , cdn isotopes ) 
 was added per l of medium , followed by shaking for 20 min .
the temperature 
 was lowered to 25 c , and iptg was added to a final concentration 
 of 1 mm .
after 14 h , cells were pelleted by centrifugation ( 5000  g , 10 min , 4 c ) and frozen at 80 c . 

cell paste ( 2 g ) was suspended in 20 
 ml of buffer a containing 1 mm pmsf .
the cells were lysed by a single 
 passage through a french pressure cell ( 14  000 psi ) .
dna was 
 precipitated by dropwise addition of 0.2 vol of buffer a containing 
 6% ( w / v ) streptomycin sulfate .
the mixture was stirred for an additional 
 10 min , and the precipitated dna was removed by centrifugation ( 20  000 
  g , 10 min , 4 c ) .
the 
 solution was stirred for an additional 20 min , and the precipitated 
 protein was isolated by centrifugation ( 20  000  g , 10 min , 4 c ) .
the pellet was dissolved in a minimal 
 volume ( 0.3 ml ) of buffer a and desalted using a sephadex g-25 column 
 ( 1  20 cm , 15 ml ) equilibrated with buffer containing bicine 
 ( 30 mm , ph 7.5 ) , tcep ( 5 mm ) , and 3% inositol .
reaction 
 buffer containing bicine ( 30 mm , ph 7.5 ) , kcl ( 30 mm ) , mgso4 ( 10 mm ) , and 3% inositol was prepared in d2o and lyophilized 
 to remove exchangeable protons , brought into the glovebox , and redissolved 
 in d2o ( 99.9% , cambridge isotope laboratories ) .
nrdd and 
 nrdg were exchanged into this buffer by repeated dilution and concentration 
 by ultrafiltration ( amicon ym-30 ) , such that < 1% h2o 
 remained .
( a ) to minimize 
 levels of formate in the assay , components of the protein storage 
 and reaction buffers found to contain a formate contaminant ( tris 
 base , glycerol , and dtt ) were substituted with compounds containing 
 undetectable levels of formate ( tea , inositol , and tcep ) .
the reaction 
 was initiated in a glovebox in a 4 c cold room by mixing a solution 
 of activated nrdd ( 20 l ) with a solution containing 5-[h]-ctp , atp , and all other components of the reaction buffer 
 ( 5 l ) .
the final reaction mixture contained nrdd ( 100 m ) , 
 5-[h]-ctp ( 1.5 mm ) , atp ( 1.5 mm ) , tea ( 30 mm , ph 7.5 ) , 
 kcl ( 30 mm ) , and mgso4 ( 10 mm ) .
after 40 s , the reaction 
 was quenched by addition of 2% perchloric acid ( 20 l ) .
( b ) nrdd was incubated with 5-[h]-ctp and atp as described 
 above , followed by addition of a mixture ( 1 ml ) of hco2na ( 10 mm ) , mgso4 ( 3 mm ) , tea ( 10 mm , ph 7.5 ) , and unlabeled 
 ctp ( 1 mm ) . subsequent to mixing , the sample was immediately quenched 
 with 17 l of 60% perchloric acid .
the total reaction time with 
 formate prior to quenching with acid was < 5 s. in both cases , 
 the samples were neutralized with koh while cooling 
 on ice , and the precipitated kclo4 was removed by centrifugation . 

quantification of 5-[h]-cyt and 5-[h]-dc was 
 carried out as described above . to a 7 ml portion of the eluate of 
 the dowex-1-borate column , containing a mixture of 5-[h]-cyt and 5-[h]-dc , was added carrier cyt and dc ( 10 
 nmol each ) .
the mixture was concentrated by lyophilization , redissolved 
 in water , cooled on ice , and the precipitated borate salts removed 
 by centrifugation .
the supernatant was analyzed by hplc using an alltech 
 econosil column ( c18 , 10 m , 250  4.6 mm ) on a waters 
 515 hplc system equipped with a 2996 photodiode array detector .
the 
 compounds were eluted with kpi ( 20 mm , ph 6.8 ) at a flow rate of 1.0 
 ml / min .
ab initio calculations were performed using the q - chem 
 software package , version 4.1 . unless 
 otherwise specified , calculations were performed in the gas phase , 
 using spin - unrestricted kohn  sham density functional theory 
 ( ks - dft ) with the b3lyp approximate functional and the 6 - 31++g * * basis set .
all spin - unrestricted calculations were verified to have spin contamination 
 of < 0.5% based on the calculated expectation of the spin operator 
 s and its deviation from the ideal value 
 of 0.75 ( for a doublet system with spin quantum number s = 0.5 ) .
visualizations were generated using the visual molecular 
 dynamics ( vmd ) program using orthographic 
 projection , with isosurface values of 0.05 for the orbitals and 0.0025 
 ( 0.05 ) for the spin densities , respectively .
the 
 thiosulfuranyl radical formed from the methanethiyl radical and ethylmethylsulfide 
 was used as a minimal model system to investigate the electronic structure 
 of the nrdd thiosulfuranyl radical .
the model was constructed using 
 the atomic coordinates of the side chains of c79 and m288 in the crystal 
 structure of bacteriophage t4 nrdd ( pdb accession i d 1hk8 , figure s1a ) .
hydrogen atoms were 
 added , and their positions optimized while fixing the heavy atoms 
 in place . a plausible reaction coordinate for the formation of a thiosulfuranyl 
 radical
at each point along the reaction profile , 
 the ( methanethiyl ) s1- ( ethylmethylsulfide ) s2 distance was fixed , and all the other coordinates 
 were allowed to relax , to obtain the potential energy of the structure 
 as a function of s1s2 separation .
the stationary points on the potential energy 
 surfaces were verified to be true minima by ensuring that they contain 
 no imaginary frequencies .
the resulting reaction coordinate represents 
 the enthalpic contribution to creating a structure of a given s1s2 separation 
 in the gas phase at 0k .
two experimental challenges 
 had to be overcome in order to study 
 the reaction of nrdd , ctp , and atp in the absence of the reductant 
 formate .
the first was to optimize g formation , and the second 
 was to remove formate contaminants in the reaction mixture components 
 so that the first half - reaction of rnr could be studied .
e. coli nrdd 
 and nrdg were expressed and purified by minimal modification 
 of reported procedures , with the inclusion 
 of 50 m zinc sulfate in the expression media for nrdd .
the 
 generation of g requires the activating enzyme nrdg , sam , 
 and a reductant for the nrdg-[4fe4s ] cluster .
previously studies have 
 shown that generation of g by nrdg can be carried out catalytically 
 with the e. coli flavodoxin ( flda)/ 
 flavodoxin reductase ( fpr ) system or with a stoichiometric amount of nrdg and photoreduced 
 deazaflavin .
we wanted to activate nrdd 
 catalytically to minimize possible interference from the activating 
 components , which are difficult to routinely remove due to the o2-sensitivity of g. we also wanted to avoid the flda / fpr 
 system because an epr signal from the flda semiquinone might complicate 
 analysis of the epr spectra .
we initially focused on using a catalytic 
 amount of nrdg activated with photoreduced deazaflavin .
however , this 
 method proved unsuccessful as this photoreducant resulted in the reversible 
 quenching of g. several photoreductant / coreductant pairs 
 were screened , including all combinations of photoreductants acriflavin , 
 fluorescein , and rose bengal and coreductants edta , tetramethylethylenediamine , 
 tea , bicine , and cysteine .
the rose bengal / bicine pair was chosen 
 because it did not reduce the nrdd - g. this pair was used for 
 all epr experiments .
however , bicine contains a small amount of formate 
 ( see removal of formate from reaction and protein 
 storage buffers section ) , and thus it was replaced with rose 
 bengal / tea for all experiments involving product analysis , where the 
 complete removal of formate is desired .
nrdd purified and activated 
 in either way had 0.5 g per peptide ( figure 2i ) and a specific activity of 1500 u / mg 
 for ctp reduction , consistent with literature values . when nrdd , [ h]-ctp , and allosteric 
 effector atp were 
 incubated in the absence of formate and analyzed for product , we were 
 surprised to detect dctp .
since the formation of dctp requires a reductant , 
 we suspected that formate contaminated one or more of our reagents . 

several methods were used to assess its presence . using a coupled 
 assay with formate dehydrogenase ,
formate was detected in the tris 
 base and glycerol used in the protein storage and reaction buffers . 

these components were thus replaced with tea and inositol . a second 
 more sensitive assay monitored [ h]-dctp formation , which 
 suggested formate presence in dtt .
we found that 
 nrdd activity could be maintained by replacing it with 5 mm tcep in 
 the nrdd storage buffer . using this formate - free buffer ,
figure 3i shows the cw x - band epr spectrum of the g that is generated 
 by nrdg .
incubation of activated nrdd with ctp and allosteric effector 
 atp for 1 min at 4 c led to the loss of g and the appearance 
 of a new radical shown in figure 3ii , accompanied 
 by a spin loss of 5% .
subsequent addition of saturating amounts 
 of formate ( 10 mm , km = 0.2 mm ) led to the disappearance of the new radical and return of the g 
 spectrum with a yield of 80% .
x - band cw epr spectra ( black ) and simulations 
 ( red ) of nrdd glycyl 
 and thiosulfuranyl radicals .
the narrow signal of the g was scaled 
 by a factor of one - tenth for comparison with the broader signals of 
 the new radical .
the spectral artifact at 337.5 mt ( ge ) is due to an impurity in the cryostat .
( ii  v ) the thiosulfuranyl radical generated 
 by reaction of ctp and atp in the absence of formate with ( ii ) nrdd 
 in h2o , ( iii ) [ -h]-cys - nrdd in h2o , ( iv ) nrdd in d2o , and ( v ) [ -h]-cys - nrdd in d2o .
in addition , three peaks at 329 , 331 , and 332 
 mt and structure in the region of 333340 mt suggest hyperfine 
 interactions with several protons . in analogy with the mechanism 
 of the class ia rnrs
, we hypothesize 
 that in the absence of formate , nrdd catalyzes cleavage of the 3 
 c  h bond and loss of h2o from ctp to form 3-keto - dctp , 
 generating a thiyl radical on c175 ( figure 1c , 7 ) . because the new radical is long - lived ( t1/2 10 min at 4 c ) ,
we propose 
 that it might be the c175 thiyl radical stabilized by a bonding interaction 
 with the conserved m382 residue on the thiyl radical loop , in the 
 form of a thiosulfuranyl radical ( figure 1c , 8) , resembling the disulfide anion radical ( figure 1b , 4 ) of the class ia rnr . in the crystal 
 structure of t4 nrdd ,
the distance between 
 the sulfur atoms of c79 and m288 is 4.4  ( figure s1a ) . to test this hypothesis , we pursued further 
 epr spectroscopy of 
 the new radical at high microwave frequency to separate g - anisotropy from hyperfine interactions , in combination with isotope 
 labeling of nrdd at x - band frequency to identify the proton hyperfine 
 interactions contributing to the spectrum .
figure 4 shows 
 the echo - detected 140 ghz epr 
 spectrum of activated nrdd reacted with ctp and atp .
the spectrum 
 is dominated by a broad signal stretching from 4.902 to 4.998 t arising 
 from a radical with a rhombic g - tensor but also shows 
 a narrow signal around 4.993 t. the narrow signal is readily assigned 
 to residual g present in the sample
the 
 principal values of its g - tensor are gx = 2.040 , gy = 2.013 , gz = 2.0016 . 

these g - values are inconsistent with a thiyl radical , 
 where the gx value is expected 
 to be shifted further downfield but are similar to g - values previously reported for putative thiosulfuranyl radicals .
140 
 ghz echo - detected epr spectrum ( black ) and simulation ( red ) 
 of the nrdd thiosulfuranyl radical in h2o .
if the new radical is partially 
 localized on c175 it will likely exhibit hyperfine interactions with 
 its -protons .
it reveals , in comparison 
 with the unlabeled nrdd ( figure 3ii ) , loss 
 of a strong hyperfine interaction , providing evidence for localization 
 of the radical on cysteine .
another , strong hyperfine interaction 
 remains and now dominates the spectrum . in search of the origin 
 of this other proton hyperfine interaction ( figure 3iii )
, we obtained the x - band spectrum of the reaction of nrdd 
 with ctp and atp carried out in d2o ( figure 3iv ) .
again , the spectrum has clearly lost a strong hyperfine 
 interaction , which suggests that the unpaired electron interacts with 
 a solvent exchangeable proton . at 4 c
this exchange is complete 
 within the 1 min required for sample preparation , and longer incubation 
 does not lead to further changes in the signal profile .
finally , 
 carrying out the reaction of [ -h]-cys - nrdd in d2o leads to a spectrum that no longer shows any resolved hyperfine 
 interactions ( figure 3v ) .
comparison of this 
 spectrum with unlabeled nrdd in d2o reveals an isotropic 
 hyperfine interaction of 30 mhz with the nonexchangeable -proton 
 of cysteine .
comparison of this spectrum with [ -h]-cys - nrdd in h2o , on the other hand , reveals a hyperfine 
 interaction of 50 mhz associated with the exchangeable proton , 
 possibly the second -proton of cysteine .
the exchange of a -proton of cysteine 
 is unexpected and thus requires further verification .
we hypothesized 
 that if this exchange involved deprotonation to form a thiyl radical 
 anion ( figure 5a ) , then introduction of a negative 
 charge near the radical could disfavor this process .
the structure 
 of t4 nrdd suggests that c175 of e. coli nrdd is positioned close to s386 , which resides on the thiyl radical 
 loop and aligns with e441 in e. coli nrda , a residue thought to act as a general base / acid catalyst in 
 the class ia rnr ( compare figures s1a and s1b ) .
we constructed the nrdd-(s386e ) mutant and reacted it with ctp 
 and atp in d2o .
1% that of 
 nrdd-(wt ) , and the new radical also formed at a much slower rate than 
 with nrdd-(wt ) , requiring a reaction time of 1 h. the x - band 
 spectrum of nrdd-(s386e ) in d2o resembles the spectrum 
 of nrdd-(wt ) in h2o ( compare figure 5bi with figure 3ii ) , which suggests that the 
 proton exchange is blocked and that the structure of the radical remains 
 largely unaffected .
( a ) proposed mechanism for proton exchange on c175 , which 
 is prevented 
 in nrdd-(s386e ) .
( b ) x - band cw epr spectra ( black ) and simulations 
 ( red ) of ( i ) nrdd-(s386e ) in d2o and ( ii ) [ -h]-cys - nrdd-(s386e ) in h2o .
the spectral artifact 
 at 337.5 mt ( ge ) is due to an 
 impurity in the cryostat .
the suppression 
 of the proton exchange by nrdd-(s386e ) allows the site of the exchangeable 
 proton to be unambiguously established . a reaction of [ -h]-cys - nrdd-(s386e ) in h2o with ctp and atp revealed 
 the spectrum in figure 5bii , which is very 
 similar to that of [ -h]-cys - nrdd-(wt ) in d2o ( figure 3v ) .
thus , the exchangeable 
 proton is a -proton of c175 , and the exchange occurs stereospecifically .
we hypothesize that the new radical involves 
 m382 ( figure 1c 7 and 8) . to test this model ,
[ -h]-met - nrdd was prepared , 
 and the resulting radical examined for the effects of isotopic substitution . 

comparison of the spectrum of [ -h]-met - nrdd ( figure 6i ) and unlabeled nrdd ( figure 3ii ) in h2o revealed sharpening of the three peaks between 
 329 and 332 mt .
moreover , in the region 333340 mt , a fine 
 structure consisting of lines separated by 0.5 mt ( marked with vertical 
 lines in figure 6i ) has become clear .
this 
 fine structure is also present in the spectrum of unlabeled nrdd in 
 h2o ( figure 3ii , see vertical lines ) 
 but is not fully resolved .
this shows that in unlabeled nrdd , the 
 fine structure is masked by a broadening of the peaks due to unresolved 
 hyperfine interactions with the three -protons of methionine .
x - band 
 cw epr spectra ( black ) and simulations ( red ) of the nrdd 
 thiosulfuranyl radical in ( i ) [ -h]-met - nrdd in 
 h2o ; ( ii ) [ -h]-met - nrdd-(s386e ) in d2o ; ( iii ) [ ,-h]-met labeled nrdd-(s386e ) 
 in h2o , and ( iv ) [ -h]-cys,[-h]-met - nrdd in h2o . see the text and table 1 for the simulation parameters . to further investigate the fine structure
, we obtained the 
 spectrum 
 of [ -h]-met - nrdd-(s386e ) in d2o ( figure 6ii ) .
d2o exchanges the envelope protons 
 around the active site , sharpening the features associated with the 
 thiosulfuranyl radical , while the s386e mutation prevents exchange 
 of the c175  proton .
comparison of this spectrum ( figure 6ii ) with that of the corresponding nrdd-(wt ) ( figure 6i ) reveals an almost identical radical signal with 
 sharpened features .
the fine structure could be reproduced in the 
 simulation by including two additional hyperfine interactions of 14 
 mhz , which we propose to be associated with the two  protons 
 of m382 .
evidence for this hypothesis was obtained from the 
 spectrum of 
 [ ,-h]-met - nrdd-(s386e ) in h2o 
 ( only the doubly labeled methionine is commercially available ) .
the 
 results shown in figure 6iii reveal the loss 
 of the two 14 mhz hyperfines , consistent with our model .
thus , 
 our studies using a combination of [ -h]-met- and 
 [ ,-h]-met - labeled proteins support the role 
 of methionine in the thiosulfuranyl radical .
as shown in figures 36 , simulations of the epr spectra 
 using the epr simulation package easyspin were successfully performed , providing the underlying g - values and hyperfine interactions that support our electronic and 
 structural assignments .
the simulation of the spectrum of nrdd-(wt ) 
 in h2o ( figure 3ii ) was built step - by - step . 

then , 
 the hyperfine interactions were estimated for each of the four different 
 types of protons from the appropriately labeled nrdds in h2o and d2o ( figure 3ii  v 
 and figure 6 ) .
finally all hyperfine interactions 
 were optimized in a manual , global fit of all acquired spectra .
the quality of all of the 9 and 140 ghz simulations improved 
 when we included an isotropic line - broadening of 0.4 mt peak 
peak 
 and a g - strain 10% of |gi - ge| , with i = x , y , z and ge the g - value for the free electron .
furthermore , all samples were found 
 to contain a residual 510% g. to simulate its contribution 
 to the spectra , its g - values ( gx = 2.0044 , gy = 2.0035 , gz = 2.0023 ) , an isotropic hyperfine interaction with a proton 
 of aiso = 39 mhz , and an isotropic line - broadening 
 1 mt peak  peak was included . for simulations of the thiosulfuranyl 
 radical on nrdd-(s386e )
, the parameters derived from the spectra of 
 nrdd-(wt ) were used , except that gx was adjusted to 2.042 .
additional observations concerning 
 the simulations require comment . 
 first , the match between our spectra and the simulations improved 
 with the introduction of a small anisotropy to three of the four proton 
 hyperfine interactions listed in table 1 .
-electron  
 and a  -proton  that lies above or below the 
 plane of the electron spin distribution ( see figure 7a and the quantum chemical calculations 
 in the quantum chemical calculations section ) . 
 because these dipolar contributions are small , we did not attempt 
 to quantify them by using more advanced fitting methods as they depend 
 on three unknown euler angles for every proton , which can not be accurately 
 determined from our current data set .
second , on the low - field 
 side of the 140 ghz spectrum ( figure 4 ) the 
 intensity is lower than predicted by the simulation . 

this is caused by anisotropy of the relaxation properties of the thiosulfuranyl 
 radical , also observed for thiyl radicals .
third , although the spectrum of [ -h]-cys - nrdd 
 in h2o ( figure 3iii ) does not show 
 any resolved fine structure due to the two -protons of methionine , 
 the quality of the simulation improves when these hyperfine interactions 
 is included .
the same is true for the spectrum of [ -h]-cys and [ -h]-met nrdd in h2o ( figure 6iv ) , where including the -proton hyperfine 
 interactions in the simulation reproduces the unresolved splitting 
 of the 334 mt peak ( marked with arrows ) .
fourth , and in contrast 
 with the above observation , in all samples 
 where the exchangeable cysteine -proton is replaced by deuterium 
 ( figures 3iv , v and 5ii ) , inclusion of the hyperfine interactions with the methionine 
 -protons in the simulation gives rise to features that are 
 not observed in the experimental spectra .
more advanced epr techniques 
 such as [ h]-endor spectroscopy are required to investigate 
 the presence of any interaction of the radical with the methionine 
 -protons in these and other samples and to more accurately 
 measure the hyperfine interactions that we have assigned . to better understand 
 the electronic structure and bonding in thiosulfuranyl 
 radicals
, we undertook a computational study using a minimal model 
 system consisting of methanethiyl radical and ethylmethylsulfide . 

dft calculations were performed to obtain a plausible reaction coordinate 
 for the formation of a thiosulfuranyl radical .
this analysis yielded 
 the reaction profile shown in figure s2 , as a function of the separation of the thiyl ( s1 ) and thioether ( s2 ) sulfur 
 atoms .
an energetically minimal structure was found at a s1s2 distance of 3.2 
  , with a binding energy of 1.8 kcal / mol relative to 
 dissociated methanethiyl radical and ethylmethylsulfide .
the 
 molecular orbitals involving the sulfur 3p orbitals of the nonoptimized 
 structure derived from the x - ray structure coordinates are shown in figure s3 , while those of the energetically minimal 
 structure are shown in figure s4 .
the unrestricted 
 dft calculation yields separate spin orbitals for the  and 
  ( up and down ) spin electrons . while all the sulfur 3p orbitals 
 of the nonoptimized structure are noninteracting ( figure s3 ) , the s1 and s2 3pz orbitals of 
 the energetically minimal structure engage in a bonding interaction 
 ( figure s4 ) .
the  * antibonding singly 
 occupied molecular orbital ( somo ) and the  bonding somo-1 are 
 highlighted in figure 7a .
together , they account 
 for a s1s2 three - electron -bond of order 1/2 .
the frontier orbitals 
 show significant electron amplitude on two of the hydrogen atoms on 
 the methanethiyl fragment .
localization of a hydrogen atom within 
 the lobe of the  * orbital could account for its lability 
 as observed in the epr experiments . the lack of bonding in the nonoptimized 
 protein structure , where s1 and s2 are separated by 4.4  suggests that 
 a conformational change is required to bring c175 and m382 within 
 bonding distance .
excess spin density coincides with the  * orbital , 
 with a small component on two of the methanethiyl hydrogens . to quantify 
 the spin excesses on an atomic level
, lwdin population analysis was performed on the spin density matrix , and 
 the resulting spin charges are shown in figure 7b .
most of the spin ( 83% ) is localized on the methanethiyl sulfur , 
 with a smaller population ( 15% ) on the thioether sulfur .
in addition , 
 a small amount of excess spin is localized on two methanethiyl hydrogens 
 ( 2.1% and 2.5% ) and to a lesser extent on several protons adjacent 
 to the thioether group ( 0.10.5% ) , which is consistent with 
 the observation of hyperfine interactions with the cysteine -protons 
 and methionine - and -protons in the nrdd radical . in 
 nrdd ,
the relative magnitude of the hyperfine interactions likely 
 depends on the angle of rotation about the cysteine and methionine 
 c  s bonds , which are dictated by the constraints of the protein 
 scaffold .
( a ) frontier molecular orbitals for the model thiosulfuranyl radical 
 formed from methanethiyl radical and ethylmethylsulfide .
diagrams 
 for the  and  spin orbitals are shown separately on 
 the left and right , respectively , together with orbital energies in 
 kcal / mol , which represent the ionization enthalpy of the electron 
 in that orbital .
for each diagram , the leftmost and rightmost orbitals 
 represent the fragment molecular spin orbital of the methanethiyl 
 and methylethylsulfide fragments , respectively , which mix to produce 
 the  bonding and  * antibonding orbitals .
snpz refers 
 to the 3pz orbital on the sulfur atom 
 of fragment n ( 1 = methanethiyl , 2 = ethylmethylsulfide ) . 

( b ) atomic spin populations obtained from lwdin population 
 analysis on the model thiosulfuranyl radical , showing excess spin 
 density localized primarily on the sulfur atoms and secondarily on 
 two of the hydrogen atoms of the methanethiyl fragment .
as shown in figure 1c , we propose that formation of the thiosulfuranyl radical involves 
 nrdd catalyzing the conversion of ctp to 3-keto - dctp ( figure 1c , 7 and 8) . to investigate 
 the fate of ctp during the reaction , the thiosulfuranyl radical
was 
 generated by incubating nrdd with 5-[h]-ctp and atp in 
 the formate - free buffer for 40 s at 4 c followed by quenching 
 in 1% perchloric acid .
workup of the reaction using a dowex-1-borate 
 column , as shown in figure 8 , gave 1.0  
 0.14 equiv of 5-[h]-cyt and 0.38  0.25 equiv of 
 dc ( identified by comigration with a standard using hplc ) .
the former 
 is a breakdown product of 3-keto - dctp ( figures 2 and 8) , consistent with our proposed 
 model , and the latter is attributed to our inability to completely 
 remove formate from the reactions components . as noted above , addition of 10 mm formate 
 to the thiosulfuranyl radical results in the recovery of 80% of the 
 initial g.
we propose that during this reaction , nrdd - bound 
 3-keto - dctp is reduced to dctp , completing the catalytic cycle . 

the experiment was repeated as described above , incubating nrdd , 5-[h]-ctp , and atp in formate - free buffer for 40 s at 4 c . 
 a 1 ml solution containing 10 mm formate and 1 mm ctp
was then added , 
 followed by immediate quenching by hand with 1% perchloric acid .
the 
 unlabeled ctp was added to dilute 5-[h]-ctp 27-fold to 
 limit the contribution of [ h]-dctp to the first turnover .
the work - up is as described ( figure 8) , 
 and the results reveal 0.12  0.03 equiv of 5-[h]-cyt 
 and 1.57  0.21 equiv of 5-[h]-dc ( see table 2 ) .
comparison with the products formed by acid quench 
 without addition of formate reflects a decrease in the amount of cyt 
 and increase in the amount of dc detected .
this suggests that 0.88 
  0.14 equiv of enzyme - bound 3-keto - dctp is consumed 
 and converted to dctp and that nrdd carries out only 0.3 additional 
 turnovers between addition of formate and acid quench . to account 
 for the 0.12 equiv of cyt remaining after reaction with 
 formate
, we suggest that during the reaction time of 40 s prior to 
 addition of formate , some amount of the 3-keto - dctp dissociates 
 from the enzyme and decomposes to cyt before or during the acid quench . 
 however , it is remarkable that the results suggest that the 3-keto - dctp 
 remains largely sequestered in the active site during this period .
products 
 formed after quenching of the thiosulfuranyl radical generated 
 by reaction of nrdd with 5-[h]-ctp and atp .
( i ) quenching 
 with perchloric acid leads to release of 5-[h]-cyt from 
 breakdown of 5-[h]-3-keto - dctp .
( ii ) addition 
 of formate prior to quench leads to conversion of 3-keto - dctp 
 to 5-[h]-dctp .
average of five 
 experiments ( results 
 for the individual trials are given in figure 
 s6 ) .
thiyl radicals have long been proposed to be involved in a diverse 
 range of enzymatic reactions from the pyruvate - ferredoxin oxidoreductase to pyruvate formate lyase and all classes of rnrs .
more 
 recent examples include mechanistically diverse glycyl radical enzymes 
 involved in fermentation , in the environmentally important 
 processes of anaerobic hydrocarbon activation , and the noncanonical 
 glycyl radical enzyme involved in the formation of methane from methyl 
 phosphonate .
however , the spectroscopic 
 observation of thiyl radicals and the demonstration of their kinetic 
 competence has been challenging due to their short lifetimes and the 
 large spin 
orbit coupling with the sulfur atom , which results 
 in short relaxation times and large g - anisotropy and 
 as a consequence broad epr lines . in the class
i and ii rnrs , three cysteines are involved in nucleotide 
 reduction ( figure 1a , b ) : the transient thiyl 
 radical located on the top face of the nucleotide and two cysteines 
 located on its bottom face that are converted to a disulfide in the 
 reduction process . only in the case of the lactobacillus 

leichmannii class ii rnr has evidence for the involvement 
 of a kinetically competent top face thiyl radical in the first half - reaction 
 ( figure 1a ) been demonstrated .
the evidence in support of this mechanism for the class i and iii 
 rnrs is inferred based on structural homology of all the classes of 
 rnrs and extensive biochemical studies . in the second half - reaction ( figure 1b ) , 
 the involvement of a thiyl radical in the nucleotide reduction by 
 the bottom face cysteines has been postulated . in the e. coli class ia 

rnr , e441 is believed to provide the proton required for the reduction 
 of the 3-keto - deoxynucleotide by the disulfide anion radical 
 via proton - coupled electron transfer ( figure 1b , 4 and 5 ) .
support for this proposal 
 has been provided by studies with the e441q mutant that causes accumulation 
 of the disulfide anion radical on the 10 s time scale , allowing its 
 detection by pulsed , high - field epr spectroscopy . however
, a limitation from the mutant studies is that the 
 trapped radical is not chemically competent for deoxynucleotide formation 
 because the essential proton for the pcet step is missing .
thus , the 
 detailed mechanism of the bottom face reduction chemistry , and specifically 
 the involvement of thiyl radicals , has remained elusive .
unlike 
 class i and ii rnrs , the class iii rnrs studied to date 
 use formate as a reductant .
we hoped 
 that examination of this rnr might shed light on the second half - reaction 
 of all rnrs .
here we have shown that omission of formate from our 
 assays leads to accumulation of a new radical upon reaction of nrdd 
 with ctp and atp , accompanied by formation of a bound cytidine species 
 proposed to be 3-keto - dctp ( figure 1c , 7 and 8) .
furthermore , addition of formate 
 converts the bound nucleotide into dctp with recovery of the g. 
 our studies suggest that the new species is a thiosulfuranyl radical 
 and demonstrate its chemical competence .
these results provide further 
 evidence for the involvement of a thiyl radical in the reductive half - reaction 
 of rnrs in general .
assignment 
 of the structure of the new radical is based on results from isotopic 
 labeling studies with various nrdds accompanied by analysis of their 
 x band epr spectra , g - values obtained by 140 ghz epr 
 studies , structural insight from the bacteriophage t4 nrdd , and computational 
 studies .
important insight is also provided by recent dft and correlated 
 ab initio calculations by van gastel et al . on the electronic structure of the cysteine thiyl radical and its 
 unusual epr parameters .
thiyl radicals exhibit broad g - anisotropy with gx - values 
 ( x axis parallel to the cs 
 bond ) that range from 2.10 to 2.49 . in 
 the cysteine thiyl
radical , the epr g - values are sensitive 
 to the energy difference between the nearly degenerate singly occupied 
 orbital ( somo with a predominant py character 
 in van gastel s system ) and one of the lone - pair orbitals ( pz ) .
this unusual 
 property makes gx highly 
 sensitive to radical conformation and h - bonds and requires an expression 
 for the g - tensor in which third - order corrections must 
 be taken into account .
the g - tensor for our new 
 radical from the 140 ghz epr spectrum is also anisotropic ( figure 4 ) , with a gx value of 2.040 .
this value is distinct from the thiyl radicals and 
 is consistent with the range of values reported for candidate thiosulfuranyl 
 radicals generated by pulse radiolysis and laser flash photolysis 
 of a range of thiols ( 2.0272.058 ) .
s bonds in these structures 
 are weak and have only been observed in small molecules containing 
 electronegative alkyl substituents ( cf3ssr2 
 or rcossr2 ) , in glasses / solid matrices , or in intramolecular cases . arguments in favor of the assignment of these 
 species as thiosulfuranyl radicals are described by symons and co - workers . in nrdd ,
the formation of the thiosulfuranyl 
 radical is likely favored by the juxtaposition of the c175 and m382 
 sulfur atoms within the active site by the protein .
our calculations 
 on a model thiosulfuranyl radical provide an explanation 
 for our observed gx value 
 relative to that of a thiyl radical .
they suggest that the  * 
 antibonding somo and the  bonding somo-1 together account for 
 a weak , s1s2 three - electron -bond of order 1/2 due 
 to the interaction between the sulfur 3pz orbital of the thiyl radical and a nonbonding orbital of the thioether . 

this interaction is expected to perturb both the symmetry and degeneracy 
 of the thiyl radical 3py and 3pz orbitals , shifting the gx value closer to that of the free electron .
our isotopic labeling studies with nrdd / ctp / atp provide strong 
 evidence for localization of the radical on cysteine and methionine 
 as hyperfine interactions associated with both cysteine -protons 
 and the methionine - and -protons are observed .
the 
 spin densities from our dft calculations also suggest that excess 
 spin of the three - electron bond includes small packets of spin localized 
 on the cysteine -protons and to a lesser extent on the adjacent 
  and  protons of methionine ( figures 7b and s5 ) .
one initially 
 puzzling observation from our epr analyses was that 
 one of the cysteine -protons on the thiosulfuranyl radical 
 was exchangeable with the solvent . however , perusal of the literature 
 focused on examining the fate of thiyl radials generated by a variety 
 of different methods in small molecules ( cysteine , penicillamine , 
 glutathione ) in d2o revealed reversible deuterium incorporation 
 into both the  and/or  positions . in cysteamine radicals generated by pulse 
 radiolysis in acidic solution , hydrogen - transfer reactions lead to 
 the equilibration of h3nch2ch2s and h3nch2ch  sh species with very fast forward and reverse rates 
 of k12  10 s and k21  1.5  10 s , respectively . in the case of nrdd c175
, we propose that its -ch bond might 
 be weakened by a hyperconjugative interaction with the singly filled 
 sulfur pz orbital , facilitating its deprotonation 
 and allowing exchange with deuterium from the solvent , although the 
 mechanism remains to be determined .
this result is also consistent 
 with our dft calculations showing the localization of a proton within 
 a lobe of the s 
s  * orbital , and the observation that 
 of the two diasteriotopic -protons on cysteine ( we do not know 
 which one ) , the one with the largest hyperfine interaction is exchanged . 

the exchange is prevented in nrdd-(s386e ) , where the negative charge 
 adjacent to the radical was designed to destabilize the conjugate 
 anion and thus disfavor deprotonation ( see figure 
 s1 ) .
finally , the rapid exchange of hydrogens adjacent to the 
 thiyl radical might provide a diagnostic for thiyl radicals in general 
 which have been , for reasons outlined above , challenging to observe .
based on earlier studies and our observations reported herein , we propose 
 the mechanistic model for nucleotide reduction shown in figure 1c for the class iii rnr .
the reaction is initiated 
 by the generation of the ctp 3-radical ( 2 ) by 
 the c384 thiyl radical . in the absence of basic residues in the active 
 site
, formate has been proposed to act as a base to catalyze water 
 loss to form a ketyl radical ( 3 ) .
the ketyl radical gains an electron from c175 and a proton 
 from formic acid , forming 3-keto - dctp and generating the thiosulfuranyl 
 radical ( 8) . for the experiments in formate - free buffer , 
 3-keto - dctp can still be generated without base catalysis . 

this observation is analogous to the e441q mutant in the class i rnr 
 where the 3-keto - dcdp is still formed , but at a much slower 
 rate .
exchange of formate between the active site and buffer 
 after formation of this stable radical rationalizes the [ h]-formate isotope effect of 2.3 measured on the reduction process .
a similar reaction between formate and dtt radicals generated 
 by pulse radiolysis has previously been reported .
the relative reduction potentials of co2 ( e = 1.90 v ) 
 and ( ch3)2co ( e = 2.10 v ) suggest 
 that reduction of 3-keto - dctp to the dctp 3-radical 
 ( 10 ) may require the concerted delivery of a proton from 
 an unknown source .
alternatively , the delivery of the proton and electron 
 may occur via a stepwise mechanism as shown in figure s7 .
reduction of the product radical generates dctp 
 and regenerates the c384 thiyl radical ( 11 ) . in 
 our mechanistic model ,
although the function of the thiosulfuranyl radical , 
 a stabilized form of the c175 thiyl radical , is not understood , its 
 stoichiometric formation and ability to catalyze the formation of 
 deoxynucleotide upon formate addition suggest its mechanistic importance . 

we hypothesize that since formate can enter and leave the active site 
 even after generation of radical intermediates , formation of this 
 species might protect the c175 thiyl radical in vivo under conditions when formate levels are low and the enzyme is saturated 
 with substrate and effector nucleotides .
the unanticipated identification 
 of a residue ( m382 ) that plays 
 a part in the reaction with formate provides us with a handle to search 
 for nrdds that use alternative reductants among the many metabolically 
 diverse organisms with sequenced genomes .
an examination of the rnrdb shows that m382 is not strictly conserved ( figure s8 ) .
all annotated archaeal and 
 bacterial nrdd sequences lacking this m382 residue contain a cysteine 
 residue in place of g383 on the thiyl radical loop adjacent to c384 
 ( figure s9 ) , in a position that may allow 
 formation of a disulfide with the bottom face thiol .
in addition , 
 the bacterial proteins contain a conserved glutamate residue aligning 
 with y542 in the active site ( compare figures 
 s1a and s9 ) , placing it in a position to act as a general base / acid 
 catalyst analogous to the role of e441 in e. coli nrda .
these residues are shown in the homology model of the thermotoga maritima nrdd given in figure s9 .
finally , archaeal nrdds , like those found in archaeoglobus veneficus and methanosarcina 
 barkerii , contain a thioredoxin - like protein in the nrddg operon .
all these observations suggest that there 
 exist nrdds that use disulfide chemistry instead of formate for nucleotide 
 reduction .
the recombinant production and characterization of some 
 of these proteins are the focus of ongoing studies .
despite the many enzymatic reactions proposed to involve thiyl 
 radicals , there have 
 been few systems that have allowed their spectroscopic and chemical 
 characterization . in this study
we found that reaction of the e. coli class iii rnr with ctp in the absence of 
 formate resulted in stoichiometric accumulation of a thiosulfuranyl 
 radical , comprising a cysteine thiyl radical stabilized by a three - electron 
 bond to a methionine residue . this new sulfur - based radical joins 
 other sulfur - based radicals observed in the other two classes of rnrs : 
 the class ia disulfide anion radical proposed 
 to be involved directly in nucleotide reduction and the class ii exchange 
 coupled
thiyl radical - cob(ii)alamin involved 
 in 3-hydrogen atom abstraction that have been experimentally 
 detected .
our results suggest that detecting thiyl radicals and controlling 
 their reactivity may require an orchestrated constellation of residues 
 adjacent to the thiyl radical that protects it from alternative chemistry 
 while allowing the reaction to proceed rapidly when the substrate 
 ( effectors ) is in the appropriate conformation .
 the 
 class iii ribonucleotide reductases ( rnrs ) are glycyl radical 
 ( g ) enzymes that provide the balanced pool of deoxynucleotides 
 required for dna synthesis and repair in many facultative and obligate 
 anaerobic bacteria and archaea . 
 unlike the class i and ii rnrs , where 
 reducing equivalents for the reaction are delivered by a redoxin ( thioredoxin , 
 glutaredoxin , or nrdh ) via a pair of conserved active site cysteines , 
 the class iii rnrs examined to date use formate as the reductant . 
 here , we report that reaction of the escherichia coli class iii rnr with ctp ( substrate ) and atp ( allosteric effector ) 
 in the absence of formate leads to loss of the g concomitant 
 with stoichiometric formation of a new radical species and a  trapped  
 
 cytidine derivative that can break down to cytosine . 
 addition of formate 
 to the new species results in recovery of 80% of the g and 
 reduction of the cytidine derivative , proposed to be 3-keto - deoxycytidine , 
 to dctp and a small amount of cytosine . 
 the structure of the new radical 
 has been identified by 9.5 and 140 ghz epr spectroscopy on isotopically 
 labeled varieties of the protein to be a thiosulfuranyl radical [ rssr2] , composed of a cysteine thiyl radical stabilized 
 by an interaction with a methionine residue . 
 the presence of a stable 
 radical species on the reaction pathway rationalizes the previously 
 reported [ 3h]-(kcat / km ) isotope effect of 2.3 with [ 3h]-formate , 
 requiring formate to exchange between the active site and solution 
 during nucleotide reduction . 
 analogies with the disulfide anion radical 
 proposed to provide the reducing equivalent to the 3-keto - deoxycytidine 
 intermediate by the class i and ii rnrs provide further evidence for 
 the involvement of thiyl radicals in the reductive half - reaction catalyzed 
 by all rnrs . 