coenzyme f420 ( 4 ) is a redox cofactor found 
 in methanogens and in various actinobacteria , while its biosynthetic 
 precursor fo ( 8-hydroxy-5-deazaflavin , 3 ) 
 can also be found in certain cyanobacteria and eukaryotes . f420 was first isolated from methanobacterium strain m.o.h . as a fluorescent cofactor involved in hydrogen metabolism 
 and in addition , m. tuberculosis , the 
 etiologic agent of tuberculosis , is predicted to contain a large , 
 yet unexplored , number of f420-dependent enzymes , some 
 implicated in nitrosative stress resistance . f420 is biosynthesized in methanocaldococcus 
 jannaschii by the action of eight enzymes with the 
 formation of the deazaflavin chromophore ( fo ) as the remaining 
 unsolved step ( figure 1 ) . despite the major biological importance of this cofactor , 
 the biosynthesis 
 of its deazaflavin core ( fo ) remains only partially understood . the formation of fo is mediated by two separate radical 
 sam active sites , one each in the cofg and cofh enzymes or both in 
 the fbic enzyme . these two radical sam domains constitute the functional 
 domains of fo synthase as we recently demonstrated . while two [ 4fe-4s ] clusters have been found 
 in other systems ( moaa , alba , hydg ) , fo synthase is an unusual multidomain radical sam enzyme 
 in that it uses two separate 5-deoxyadenosyl radicals to catalyze 
 fo formation . the 
 structure of f420 shown 
 contains a single glutamic acid , which is designated as f420 - 1 . we recently reconstituted the fo synthase and 
 identified 
 diaminouracil ( 5-amino-6-ribitylamino-2,4(1h,3h)-pyrimidine - dione ) 2 and tyrosine 1 as the enzyme substrates . the detection 
 of diaminouracil 2 bound to freshly purified cofh suggested 
 that cofh catalyzes the early steps in deazaflavin formation . adg and adh correspond to the 5-deoxyadenosyl 
 radicals generated at the cofg and the cofh active sites by reduction 
 of sam using the active site [ 4fe-4s ] cluster . abstraction of the tyrosine amine hydrogen atom by the cofh 5-deoxyadenosyl 
 radical gives 5 , which then undergoes fragmentation leading 
 to the formation of the p - hydroxybenzyl radical 7 . this then undergoes addition to the double bond of diaminouracil 2 followed by oxidation to give 9 . transfer of 9 from the cofh active site to the cofg active site gives 10 . cyclization to 13 , followed 
 by oxidation by the [ 4fe-4s ] cluster and elimination 
 of ammonia completes the formation of the deazaflavin 3 . the tyrosine lyase activity of 
 fo synthase is likely 
 to be mechanistically analogous to the tyrosine lyase activity of 
 hydg ( involved in [ fefe]-hydrogenase maturation ) and thih ( involved in anaerobic thiamin thiazole biosynthesis ) . a recent structure of tryptophan lyase ( nosl ) 
 demonstrated that the initial hydrogen atom abstraction was occurring 
 from the amine nh , suggesting that the 
 corresponding tyrosine lyases are also likely to occur by hydrogen 
 atom abstraction from the amino group rather than the previously assumed 
 phenolic hydroxyl . based on these data , we elaborated the mechanism 
 shown in figure 2 as a starting point for our 
 mechanistic investigations on fo synthase . in this 
 paper , we report a detailed mechanistic study on this complex 
 enzyme that led us to identify ( 1 ) the hydrogen atoms abstracted from 
 the substrate by the two radical sam domains , ( 2 ) the second tyrosine - derived 
 product , ( 3 ) the reaction product of the cofh - catalyzed reaction , 
 ( 4 ) the demonstration that this product is a substrate for cofg , and 
 ( 5 ) a stereochemical study that is consistent with the formation of 
 a p - hydroxybenzyl radical at the cofh active site . our study elucidates a new catalytic motif in radical sam enzymology 
 involving the use of two 5-deoxyadenosyl radicals to mediate 
 the formation of a complex heterocycle . reconstitution 
 of the cofg / cofh - catalyzed reaction in the presence 
 of d7-tyrosine resulted in the incorporation of a single 
 deuterium atom into 5-deoxyadenosine as judged by mass spectrometry 
 ( ms ) analysis , which showed an increase in the intensity of the 253.1 
 peak relative to the 252.1 peak ( 48.6% vs 13.4% in the reaction performed 
 with d7-tyrosine compared with unlabeled substrate , respectively , 
 figure 3a , b , left panel ) . ms analysis of the 
 fo formed from d7-tyr showed that it contained 
 four deuteriums ( figure 3b , right panel ) . reconstitution 
 of the cofg / cofh reaction with other isotopologues of tyrosine ( [ 2-d1]- , [ 3,3-d2]- , [ 2,6-d2]- , and [ 3,5-d2]-tyr ) 
 localized the site of deuterium abstraction to the -carbon 
 of tyr since deuterium incorporation in 5-deoxyadenosine was 
 detected only when using d7- or 3,3-d2-tyr as 
 substrate ( figure 3c ) . ms analysis of 
 the fo formed in the reaction with [ 3,3-d2]-tyr 
 demonstrated that the second -deuterium was retained in fo . using [ 3,5-d2]-tyr led to the 
 incorporation of both deuterium atoms into fo while only 
 one was incorporated when using [ 2,6-d2]-tyr as substrate ( supplementary 
 figure 1 ) . conversely , no deuterium incorporation into the 
 products was observed with [ 2-d1]-tyr ( supplementary figure 1 ) . this confirmed that , in addition 
 to one deuterium on the 3-position , a deuterium from a second nonexchangeable 
 site ( i.e. , 2/6 position ) is removed . similar results 
 were obtained using the full length fbic enzyme demonstrating that 
 both types of fo synthase use an identical mechanism . deuterium 
 incorporation into 5-deoxyadenosine ( left spectrum , 
 [ m + h ] = 252.1 da ) and fo ( right spectrum , 
 [ m + h ] = 364.1 da , [ m + k ] = 402.1 da ) from 
 various isotopologues of tyrosine : ( a ) tyrosine , ( b ) d7-tyrosine , or ( c ) 3,3-d2-tyrosine . the percentages written 
 above the 253.1 da peak indicate the relative height compared to the 
 252.1 peak . each reaction mixture contained methyl viologen , sam , 
 diaminouracil , dn - tyrosine , cofh , and 
 cofg . the observation that deuterium 
 transfer from a second site on stably 
 labeled tyrosine to the 5-deoxyadenosyl radical was not occurring 
 suggested that the second hydrogen atom transfer was from an exchangeable 
 site . this was confirmed by demonstrating the incorporation of deuterium 
 from solvent into 5-deoxyadenosine when the cofh reaction 
 was run in a buffer containing 80% d2o . in addition , noticeable 
 peaks corresponding to [ 5,5-d2]-5-deoxyadenosine 
 and [ 5,5,5-d3]-5-deoxyadenosine 
 were observed in the mass spectrum ( figure 4 ) . the residual 252.1 peak in spectrum a is most likely due to the 
 uncoupled formation of 5-deoxyadenosine and to the presence 
 of h2o in the reaction mixture . cofh - catalyzed deuterium 
 incorporation into 5-deoxyadenosine 
 in reactions run in 80% d2o containing sam , diaminouracil 
 and methyl viologen . ( a ) reaction run in the presence of tyrosine 
 showing an enhanced 253.1 peak ( calculated to be 69.4 , 21.5 and 16.7% 
 for the [ m + d + h ] , [ m + d2 + h ] , [ m + d3 + h ] , respectively , in panel a and 
 30.8% for the [ m + d + h ] in panel b ) . ( b ) reaction run 
 in the absence of tyrosine showing the uncoupled production of 5-deoxyadenosine . 5-deoxyadenosine has an expected [ m + h ] of 252.1 m / z with the 253.1 peak calculated to be 
 12.9  3% . glyoxylate , derived from 
 the hydrolysis of the glycine imine 6 , was the most likely 
 second product resulting from the fragmentation 
 of the tyrosyl radical 5 . our initial attempts to trap 
 glyoxylate as the 2-quinoxalinol 17 , by treating the 
 reaction mixture with o - phenylenediamine , failed because the glycerol used to stabilize the enzyme contained 
 relatively large quantities of glyoxylate as an impurity . we therefore 
 performed the reaction using [ n , c9]-tyr , which allowed for the selective lc - ms detection and quantitation 
 of enzymatically produced glyoxylate ( figure 5 ) . we were able to detect glyoxylate only in 
 the presence of cofh ( figure 5 ) , further supporting 
 the proposal that cofh is the enzyme responsible for the tyrosine 
 cleavage reaction . ( a ) 
 proposed scheme for the generation of glyoxylate 15 by 
 hydrolysis of the glycine imine 6 and its trapping using o - phenylenediamine 16 . ( b ) extracted ion chromatograms 
 at m / z 148.05  0.1 of labeled 
 2-quinoxalinol 17 generated from a reaction mixture containing 
 cofg + cofh + sam + sodium dithionite + [ n , c9]-tyrosine + 2 ( red trace ) ; cofh + sam 
 + sodium dithionite + [ n , c9]-tyrosine 
 + 2 ( blue trace ) . control reactions lacking sam , sodium 
 dithionite , tyrosine , or cofh did not produce labeled glyoxylate . hplc analysis 
 of the cofh reaction mixture revealed the formation of a peak eluting 
 at 11.2 min . this peak appeared only in reaction mixtures containing 
 cofh , 1 , 2 , sam and reduced methyl viologen 
 ( or dithionite ) ( figure 6 ) . lc - ms analysis 
 yielded a protonated molecular ion at 383.1573 m / z when the reaction was reconstituted in the presence of 
 tyrosine and 390.1775 m / z when the 
 reaction was reconstituted in the presence of [ n , c9]-tyr ( figure 7 ) . this 
 demonstrated that cofh catalyzed the formation of a product containing 
 seven tyrosine - derived carbons and suggested a molecular formula of 
 c16h22n4o7 ( [ m + h ] calcd 383.1561 , 3.3 ppm error ) . cid fragmentation of the 
 unlabeled product resulted in the formation of product ions at m / z 107.1 and 277.1 , while the labeled 
 product showed product ions at m / z 114.1 and 277.1 ( figure 7 ) . this suggested 
 that the smaller fragment contained all the tyrosine - derived carbon 
 atoms ( i.e. , 7 carbon atoms ) while the 277.1 fragment originated from 
 diaminouracil . ( b ) hplc chromatogram of the reaction mixture containing 
 cofh + sam + methyl viologen ( reduced ) + 1 + 2 ( red ) showing a new product eluting after 11.15 min . reaction mixtures 
 lacking reduced methyl viologen ( green ) , sam ( black ) , or tyrosine 
 ( blue ) did not show this product . ( a ) mass spectrum of 
 the product generated from tyrosine ( [ m + h ] obs . 383.1573 , 
 calcd for 9 , 383.1561 , 3.3 ppm error , [ m + k ] obs . 421.1132 , calcd for 9 , 421.1120 , 2.8 ppm error ; 
 ( b ) mass spectrum of the product generated from [ n , c9]-tyr ( [ m + h ] obs . 390.1775 , calcd 
 for [ c7]-9 , 390.1796 , 5.4 ppm 
 error , [ m + k ] obs . 428.1322 , calcd for 9 , 428.1355 , 7.7 ppm error ) ; ( c ) ms of the product from 
 tyrosine ( m / z 383.1 ) ; ( d ) ms of the product from [ n , c9]-tyrosine ( m / z 390.1 ) . the isolation 
 width was 10 m / z and the collision 
 energy was 30 v. ( e ) proposed cid fragmentation pattern of the cofh 
 product 9 . the cofh reaction product was then produced on 
 a larger scale , 
 purified by hplc and analyzed by nuclear magnetic resonance ( nmr ) 
 spectroscopy . the h nmr spectrum had doublets at 6.77 
 and 6.57 ppm , consistent with a para - substituted benzene ring with 
 electron - donating substituents , multiple signals in the 33.8 
 ppm region consistent with protons attached to oxygen - bound carbons , 
 and two doublets at 2.87 ppm suggestive of a benzylic methylene group . using 2d nmr techniques ( h  h cosy , h  c hsqc , and h  c hmbc ) , the cofh reaction product was unambiguously identified 
 as compound 9 ( supplementary figures 
 210 ) . to confirm that 9 was an intermediate 
 and not a shunt 
 or a decomposition product , we produced d2 - 9 on a large scale using cofh and [ 3,3-d2]-tyr . treatment 
 of the hplc purified compound with reduced cofg resulted in the formation 
 of fo . ms analysis of the reaction products demonstrated 
 deuterium incorporation into 5-deoxyadenosine and fo ( figure 8) . the mixture of m / z 364/365 in the mass spectrum of fo is due to the nonenzymatic reduction of [ 5-d1]-fo by dithionite followed by nonenzymatic aerobic oxidation 
 after removal of the reaction mixture from the anaerobic chamber as 
 was previously observed during the initial characterization of f420/f0 . incorporation of deuterium 
 into 5-deoxyadenosine in reactions 
 consisting of sam , [ 5,5-d2]-9 and dithionite 
 reduced cofg . ( a ) mass spectrum of 5-deoxyadenosine ( calcd 
 252.1 with a 253.1 peak calculated to be 12.9  3% relative intensity ; 
 ( b ) mass spectrum of fo ( calcd 364.1 ) . the presence of 
 the 252.1 peak in the 5-deoxyadenosine ms is most likely due 
 to the uncoupled production of 5-deoxyadenosine . disruption of the conserved cxxxcxxc motifs 
 of fbic individually 
 resulted in the variants fbic - c1 ( cluster is disrupted in the cofg 
 homologous domain ) and fbic - c2 ( cluster is disrupted in the cofh homologous 
 domain ) . both protein variants had all three cysteine residues substituted 
 with alanine residues , thereby abolishing the [ 4fe-4s ] cluster . incubation 
 of each variant with tyrosine , diaminouracil , sam , and flavodoxin / flavodoxin 
 reductase led to the identification of a new peak only in the fbic - c1 
 reaction ( figure 9 ) . the intermediate was subjected 
 to lc - ms / ms analysis and had an identical retention time , protonated 
 ion and ms fragmentation pattern to intermediate 9 generated by cofh . ( a ) hplc analysis 
 ( abs 257 nm ) of the reaction mixtures containing sam + tyrosine ( 1 ) + diaminouracil ( 2 ) + flavodoxin / flavodoxin 
 reductase in the presence of fbic ( wild - type ) ( 25 m ) or variants 
 ( fbic - c1 or fbic - c2 , each at 50 m ) . ( b ) hplc analysis ( abs 
 257 nm ) of the reaction of fbic - c1 demonstrating that the production 
 of the intermediate is dependent on both tyrosine ( 1 ) 
 and diaminouracil ( 2 ) . mixing of fbic - c1 and fbic - c2 resulted in the formation of 
 fo ( figure 10 ) demonstrating that 
 both 
 variants contain a successfully reconstituted [ 4fe-4s ] cluster and 
 are fully active . interestingly , this confirmed that a stable diffusible 
 reaction intermediate is released from one radical sam domain ( cofh 
 or the c - terminal part of fbic ) to the other one ( cofg or the n - terminal 
 part of fbic ) . surprisingly , the rate of product formation for the 
 wild - type enzyme ( 25 m enzyme = 50 m [ 4fe-4s ] ) was similar 
 to the rate of product formation for the mixture of the two mutated 
 enzymes ( 50 m each = 50 m [ 4fe-4s ] ) . this suggests that 
 fbic catalyzes two independent reactions where intermediate 9 is not transferred directly from the c - terminal domain to 
 the n - terminal domain but most likely diffuses from one active site 
 to the other . fo production by fbic - c1 and fbic - c2 variants 
 compared 
 to fbic - wt . the fbic proteins were incubated with sam , tyrosine ( 1 ) and diaminouracil ( 2 ) , and the reaction was 
 initiated with the flavodoxin / flavodoxin reductase system . the estimated 
 rate of fbic - catalyzed fo formation is 2.4  10 s. ms analysis of the product generated by treating [ 2,3-d2 , 3s]-tyr or [ 3-d , 3r]-tyr with 
 reduced cofg / cofh demonstrated the formation of deazaflavin as a 1:1 
 mixture of fo and d1-fo . this product 
 ratio was independent of the stereochemistry at c3 of the starting 
 tyrosine ( figure 11b , c ) demonstrating that 
 the stereochemical information at this carbon is lost during the formation 
 of fo . a control experiment using [ 3,3-d2]-tyr 
 demonstrated that fo reduction / oxidation was not occurring 
 under the reaction conditions ( figure 11a ) . fo synthase catalyzes the reductive 
 condensation of 
 tyrosine 1 and ribityl - diaminouracil 2 in 
 a reaction catalyzed by two radical sam enzymes ( cofg and cofh ) or 
 the two - domain enzyme fibc . the central 
 mechanistic question for this reaction is how two highly reactive 
 5-deoxyadenosyl radicals cooperate to form the deazaflavin 
 chromophore . , we report the isolation and characterization 
 of the cofh reaction product and describe a series of experiments 
 that enable us to test this mechanistic hypothesis . stereochemistry of deuterium 
 transfer from c3 of tyrosine during 
 cofg / cofh - catalyzed fo formation . ( a ) mass spectrum of 
 fo derived from [ 3,3-d2]-tyr ; ( b ) mass spectrum 
 of fo derived from [ 2,3-d2 , 3s]-tyr ( 1c ) ; ( c ) mass spectrum of fo derived 
 from [ 3-d , 3r]-tyr ( 1d ) . ( fo calcd [ mh + h ] 364.1 , calcd [ md + h ] 365.1 , calcd [ mh + k ] 402.1 , 
 calcd [ md + k ] 403.1 . most radical sam enzymes use the 5-deoxyadenosyl radical 
 to abstract a hydrogen atom from the substrate ( dph2 and mqne are exceptions ) . since fo synthase uses two 5-deoxyadenosyl radicals , 
 two hydrogen atom abstractions are likely to occur during fo formation . we determined that one of these takes place at the c3 
 position of tyrosine by characterizing the 5-deoxyadenosine 
 produced in the cofg / cofh reaction using various deuterated tyrosine 
 isotopologues ( figure 3 ) . this experiment also 
 demonstrated the loss of a single deuterium in the fo produced 
 from [ 2,6-d2]-tyr ( supplementary figure 1d ) as expected due to c failure to observe 
 a second hydrogen atom transfer from the stable 
 deuterated tyrosine isotopologues suggested that the second hydrogen 
 atom abstraction was occurring from an exchangeable position on tyrosine 1 or diaminouracil 2 . consistent with this prediction , 
 treatment of unlabeled substrates 1 and 2 with cofh in d2o buffer resulted in deuterium transfer 
 to 5-deoxyadenosine ( figure 4 ) . phylogenetic 
 analysis of hydg , thih , cofh , and cofg with clustal omega shows that cofh clusters with thih and hydg 
 with cofg present as an out - group ( supplementary 
 figure 11 ) . this sequence similarity between cofh , hydg , and 
 thih suggested that these enzymes might catalyze a similar tyrosine 
 fragmentation ; hence we initially proposed that the second hydrogen 
 atom abstraction is occurring from either the phenolic hydroxyl or 
 the amino group of tyrosine ( figure 12 ) . since 
 cofh catalyzes the formation of intermediate 9 , generation 
 of the radical at the exchangeable site must precede generation of 
 the radical at the nonexchangeable site . this is consistent with the 
 proposed order of the hydrogen atom abstraction events shown in figure 2 . we also observed significant [ m + 2 ] and [ m + 
 3 ] peaks for the 5-deoxyadenosine produced in the cofh reaction 
 ( figure 4 ) . this observation suggests that 
 the hydrogen atom abstraction from the exchangeable site is reversible 
 ( 1 to 5 in figure 2 ) and establishes that the rate of the back hydrogen atom transfer 
 is competitive with the rate of the -scission reaction . the 
 relevant bond dissociation energies of phenol , methylamine and 5-deoxyadenosine 
 are 362 , 425 , and 433 kj mol , respectively . these thermochemical data support hydrogen atom 
 abstraction from the amine nh rather than from the phenolic oh because 
 the phenoxy radical is not sufficiently reactive to abstract a hydrogen 
 atom from 5-deoxyadenosine . the reversibility also eliminates 
 the possibility of a protein glycyl radical at the cofh active site 
 because the bond dissociation energy of glycine is 358 kj mol demonstrating that the glycyl radical is also insufficiently 
 reactive to abstract a hydrogen atom from 5-deoxyadenosine . our mechanism predicts that the tyrosine 
 radical 5 will fragment to form glycine imine 6 which should 
 then undergo hydrolysis to glyoxylate 15 ( figure 5a ) . our search strategy for this putative product 
 involved its conversion to a stable chromophoric 2-quinoxalinol 17 by derivatization with o - phenylenediamine 16 followed by detection and quantitation using lc - ms . the 
 enzymatic reaction was run using [ n , c9]-tyr , and the resulting [ 2,3-c]-2-quinoxalinol 
 could be unambiguously differentiated from the unlabeled contaminant . 
 in this way , we demonstrated that the amount of glyoxylate formed 
 averaged 1.1 times the amount of fo formed in two separate 
 experiments . a similar amount of glyoxylate was present in the cofg / cofh 
 coupled reaction and the cofh only reaction ( figure 5 ) , demonstrating that glycine imine formation is catalyzed 
 by cofh . comparison of the reaction catalyzed by thih ( thiamin biosynthesis ) 
 hydg ( fefe hydrogenase biosynthesis ) , nosl ( nosiheptide biosynthesis ) , 
 and the proposed reaction catalyzed by cofh ( f420 biosynthesis ) . reversible abstraction of the exchangeable hydrogen atom has been 
 observed for thih , ( begley , t . we previously demonstrated that 
 cofh generated a product that served 
 as a substrate for cofg . here we characterized 
 this product ( figure 6 ) and determined its 
 structure as compound 9 . to demonstrate that this compound 
 was an intermediate , it was enzymatically synthesized on a large scale 
 using [ 3,3-d2]-tyr , purified by hplc and then treated with 
 dithionite reduced cofg and sam . analysis of the reaction by lc - ms 
 showed cofg - dependent production of fo and also showed 
 the incorporation of deuterium into enzymatically generated 5-deoxyadenosine 
 and fo ( figure 8) . in this experiment we also observed washout of deuterium from [ d1]-fo , presumably due to its reduction by dithionite followed by aerobic 
 oxidation with loss of deuterium . in a complementary experiment , 
 we created two variants of fbic 
 in which either the first or second cxxxcxxc motif was disrupted by 
 alanine substitution ( named fbic - c1 or fbic - c2 , respectively ) . when 
 the two variants were incubated with substrates , we observed a new 
 peak only in the reaction mixture containing fbic - c1 ( figure 9 ) . this was identified as compound 9 by co - elution with an authentic standard , molecular mass determination 
 and ms fragmentation . we then demonstrated that fbic - c2 
 could also convert 9 to fo and that the rate 
 of fo production by wild - type fbic was approximately equal 
 to the rate of fo production by a fbic - c1/fbic - c2 mixture 
 ( figure 10 ) . this suggests that fbic catalyzes 
 two independent reactions where intermediate 9 is not 
 transferred directly from the c - terminal domain to the n - terminal 
 domain but instead follows diffusion from one active site to the other . our mechanism is in line with recent epr results obtained with 
 hydg , which show evidence for the formation of a dehydroglycine and p - hydroxylbenzyl radical during tyrosine c-c 
 bond cleavage . our initial attempts to 
 trap the proposed p - hydroxybenzyl radical 7 were unsuccessful because cofh did not catalyze the tyrosine lyase 
 reaction in the absence of diaminouracil 2 . analysis 
 of the stereochemistry of the c  c bond formation leading to 9 , using tyrosine made chiral at c3 by deuterium substitution , 
 suggested an alternative approach for the detection of 7 ( figure 13 ) . in this approach , 3-deuterio 
 tyrosine , chiral at c2 and c3 , would 
 generate the protein - bound deuterated p - hydroxybenzyl radical 7a . we propose that this radical intermediate could scramble the stereochemical 
 information originally present at c3 of tyrosine by c  c bond 
 rotation or by flipping of the entire radical . it is well established 
 that the ring of tyrosine and phenylalanine can undergo rapid flipping 
 in the interior of a protein . radical addition to 2 followed by oxidation would then give a mixture of 9a and 9b , the deuterated epimers of 9 . stereospecific hydrogen atom abstraction by the 5-deoxyadenosyl 
 radical at the active site of cofg would give 11 and 11a which would be converted to a mixture of 3 and 3a as shown in figure 2 . if 7 is not an intermediate , this mechanism of stereochemical 
 scrambling could not operate , and the deazaflavin formed would be 
 exclusively protonated or deuterated at c5 depending on the stereochemistry 
 of the starting tyrosine . to test this , [ 2,3-d2 , 3(s)]- and [ 3(r)-d1]-tyr lc - ms analysis demonstrated that the fo produced 
 was a 1:1 mixture of 3 and 3a consistent 
 with the intermediacy of the p - hydroxybenzyl radical 7 . in this paper we describe a set of experiments that 
 explain how 
 two 5-deoxyadenosyl radicals cooperate to assemble the deazaflavin 
 chromophore of the f420 cofactor . in this mechanism , abstraction of the tyrosine amine hydrogen atom 
 by the cofh 5-deoxyadenosyl radical gives 5 , 
 which then undergoes fragmentation leading to the formation of the p - hydroxybenzyl radical 7 . addition of this 
 radical to diaminouracil 2 followed by oxidation gives 
 intermediate 9 . this intermediate diffuses to the cofg 
 active site where a second hydrogen atom abstraction generates 11 . cyclization to 13 followed by oxidation and 
 elimination of ammonia completes the formation of the deazaflavin 3 . this mechanism is consistent with the copurification 
 of 2 with cofh , with the reversibility of the first hydrogen 
 atom transfer 
 ( 1 to 5 ) , with the second hydrogen atom 
 abstraction occurring from c3 of tyrosine ( 10 to 11 ) and with the formation of the glycine imine 6 as a byproduct . the scrambling of stereochemistry at the c3 of tyrosine 
 during fo formation also supports the formation of the p - hydroxybenzyl radical 7 . finally , it was 
 possible to trap 9 , the product of the cofh - catalyzed 
 reaction , and to demonstrate that it was a substrate for cofg . finally , this paper reveals how a major cofactor of the prokaryotic 
 world is assembled by an unprecedented mechanism and provides yet 
 another example of the remarkably complex chemistry that nature uses 
 in the assembly of the heterocyclic intermediates used in cofactor 
 biosynthesis . strategy to detect  bond cleavage 
 involving loss of stereochemical 
 information at c3 of tyrosine .
coenzyme f420 is a redox cofactor found in methanogens 
 and in various actinobacteria . despite the major biological importance 
 of this cofactor , 
 the biosynthesis of its deazaflavin core ( 8-hydroxy-5-deazaflavin , 
 fo ) is still poorly understood . 
 fo synthase , 
 the enzyme involved , is an unusual multidomain radical sam enzyme 
 that uses two separate 5-deoxyadenosyl radicals to catalyze 
 fo formation . in this paper , we report a detailed mechanistic 
 study on this complex enzyme that led us to identify ( 1 ) the hydrogen 
 atoms abstracted from the substrate by the two radical sam domains , 
 ( 2 ) the second tyrosine - derived product , ( 3 ) the reaction product 
 of the cofh - catalyzed reaction , ( 4 ) the demonstration that this product 
 is a substrate for cofg , and ( 5 ) a stereochemical study that is consistent 
 with the formation of a p - hydroxybenzyl radical at 
 the cofh active site . 
 these results enable us to propose a mechanism 
 for fo synthase and uncover a new catalytic motif in radical 
 sam enzymology involving the use of two 5-deoxyadenosyl radicals 
 to mediate the formation of a complex heterocycle .