e. coli alkb is a member of the fe and -kg dependent 
 dioxygenase superfamily of enzymes .
alkb 
 can repair alkylated bases such as 1-mea and n - methyl 
 cytosine ( 3-mec ) via an oxidative dealkylation .
the proposed mechanism , based on the mechanism of the related 
 enzyme taud , involves a series of steps 
 that can be separated in two parts . the first part is composed of 
 the formation of an fe = o ( ferryl ) intermediate 
 along with the release of co2 and formation of succinate . 

after the formation of the iron(iv)oxo , the oxo moiety undergoes 
 a reorientation from an axial to an equatorial position .
the subsequent 
 steps comprise the second part , which involve the oxidation of the 
 methyl moiety on the base as shown in scheme 1 ( see supporting information ( si ) scheme s1 for the full mechanism including part 1 ) . after the reorientation of 
 the oxo , the fe = o 
 moiety abstracts a hydrogen atom from the methyl group of 1-mea , followed 
 by the oh rebound to the carbon radical .
subsequently , the proton 
 on the recently added oh is transferred and the c 
n bond breaks , 
 resulting in the formation of formaldehyde . however , the details of 
 the formation of formaldehyde , such as where the proton is transferred 
 and when the c 
n bond breaks , are still not clear . moreover , 
 recent experimental discoveries suggest a possible alternative pathway . 
 the crystal structure of an intermediate in the dealkylation of 3-mec 
 has been recently reported .
based on the crystal data and qm / mm calculations , 
 a zwitterion structure was proposed . in 
 addition , time - resolved raman spectra reveal the possible existence 
 of a metal - coordinated oxygenated intermediate , such as fe  o 
c for taud , another dioxygenase undergoing a similar 
 mechanism to alkb . in the case of 
 1-mea as substrate , the zwitterion structure and 
 fe  o  c complex may form after the deprotonation 
 of the product of the oh rebound process .
the crystal structures of 
 alkb with succinate and different substrates ( pdb i d : 2fdg , 2fdj , 3ois , 3oiu , 3oit , 3oiv ) show a vacancy 
 between the succinate and the aspartate residue ( asp133 ) bound to 
 iron ( see figure 1 for 2fdg ) .
this vacancy is a result of the reorientation of the 
 oxo moiety from an equatorial to an axial position .
this 
 vacancy can be occupied by a water molecule , which results in the 
 traditional pathway as shown in scheme 2 ( h2o pathway ) . on the other hand ,
the physiological ph is slightly 
 basic , and the optimal ph for repair of 1-mea is 7.58 .
moreover , hydroxide carries a negative charge 
 and is a stronger iron - binding ligand than water .
hence , an alternative 
 pathway ( oh pathway ) with the participation of 
 hydroxide is possible ( see scheme 3 ) .
active site of alkb with 1-mea in the 
 crystal structure ( pdb i d : 2fdg ) .
furthermore , the relative 
 positions of the ch2oh ( or ch2o ) moiety connected to the 
 dna substrate with respect to the iron atom are not the same for different 
 dna bases in their corresponding crystal structures ( see si figure s1 ) . in some crystal structures ( 3oiu )
, the substrates 
 are bound to the iron center while for others ( 3ois , 3oiv ) the oxidized methyls 
 are unbound , and in another case , the moiety is located in an intermediate 
 position ( 3oit ) . in the case
when the substrate is unbound from the metal center , 
 the vacancy on the iron may be occupied by a water molecule .
we have previously used ab initio qm / mm to study the rate - limiting 
 h atom abstraction step for the h2o pathway in detail . in this contribution , we elucidate the h2o pathway ( scheme 2 ) after the hydrogen 
 abstraction step , which includes the oh rebound step and the formation 
 of formaldehyde based on the results from qm / mm simulations . in addition , 
 we report results from qm / mm simulations of the new oh pathway ( scheme 3 ) and its comparison with 
 the h2o pathway to provide new theoretical insights and 
 their comparison to previous experimental findings . in section 2
, we present the details for the setup of the systems 
 including the required structures for the different steps and the 
 computational methods .
subsequently , a detailed analysis of the results 
 for the two different pathways explored , h2o and oh , is presented .
the computational methods and structure preparation follow our 
 previous study . in brief , after adding hydrogen atoms , water box , 
 and counterions to a crystal structure of alkb ( pdb i d : 2fdg(5 ) ) , we carried out molecular dynamics ( md ) simulations in 
 the nvt ( canonical ) ensemble at 300 k using the amberff99 
 force field with a 1 fs step size with an 8  cutoff for nonbonded 
 interactions and particle mesh ewald to treat long - range coulomb interactions .
the snapshot with 
 the lowest qm / mm energy among ten selected snapshots was chosen for 
 further optimization on all reactants , intermediates , and products . 

the qm / mm calculations were performed with an in - house program that 
 links a modified version of gaussian09 with a modified version of tinker .
all qm / mm optimizations were performed using the iterative method 
 proposed by zhang et al . using the electrostatic embedding scheme .
the pseudo - bond approach was used to model the 
 boundary atoms at the qm / mm interface .
the tss were optimized using the qst3 method starting with the structure 
 that has the highest energy obtained from the optimized paths calculated 
 with the quadratic string method .
the 
 water coordinated to the iron was replaced by oh for the oh pathway without running md simulations 
 before the qm / mm optimizations . following the results from our previous 
 simulations ,
we have employed the b97xd functional coupled to the 6 - 31g(d , p ) basis set for the 
 qm part .
the structures of reactants , intermediates , and products 
 were confirmed to have no imaginary frequency and all transition states 
 ( ts ) only have one imaginary frequency corresponding to the vibration 
 along the reaction coordinate connecting the two minima for that step . to understand the interactions between fe and its surrounding ligands , 
 noncovalent interaction ( nci ) analysis 
 was performed .
nci analysis plots the reduced density gradient versus 
 the product of the sign of the second eigenvalue ( 2 ) of the 
 electron - density hessian matrix and the electron density . in practice , 
 for visualization purposes , a chosen ( small ) reduced electron gradient 

the types of the interactions 
 can be distinguished by the sign of 2 and represented by the 
 different colors .
positive 2 means repulsion while negative 
 2 means attraction . for the color scale ( blue / green / red ) , consistent 
 with the original nci convention , red represents repulsion , and blue 
 represents attraction .
the value of the electron density is represented 
 by the depth of the color .
a deeper color means larger electron density , 
 and small electron density is green .
therefore , a red surface indicates 
 relatively strong repulsion ; a blue surface represents relatively 
 strong attraction ; and a green surface is a sign of relatively weak 
 interaction .
this analysis has been proven to be a powerful tool to 
 probe the interactions in small - size molecules and large - size 
 systems such as enzymes .
in this section , we 
 present the results and discussion for the 
 steps after the formation of fe 
section 3.1 presents 
 the comparison of the hydrogen abstraction step between the oh and h2o pathways regarding the energetics 
 and the electronic structure of fe  oxo . the differences 
 between these two pathways on the oh rebound step and the unbinding 
 of the dna base from the metal and the formation of formaldehyde step 
 are discussed in sections 3.2 and 3.3 , respectively . finally , in section 3.4 , we present the experimental findings based on 
 the complete energetic picture for these two pathways .
our previous study on the 
 hydrogen abstraction step of the h2o pathway shows there 
 are two substates for each spin state as fe 
oxo 
 tends to be fe  oxyl where one electron is transferred 
 from fe to oxo . for the total quintet spin state ,
oaf , where s = 5/2 ( high spin , hs ) fe is antiferromagnetically 
 ( af ) coupled with s = 1/2 o. the second state is fe  of where s = 3/2 ( intermediate 
 spin , is ) fe is ferromagnetically coupled with s = 1/2 o. similarly , 
 for the total triplet spin state , there are also two substates : fe  oaf where s = 3/2 ( intermediate 
 spin , is ) fe is antiferromagnetically ( af ) coupled with s = 1/2 
 o and fe  of where s = 
 1/2 ( low spin , ls ) fe is ferromagnetically coupled with s = 1/2 o 
 ( see si figure s2 for the electronic configuration 
 diagram ) .
figure 2 depicts the relative energies for the hydrogen abstraction step 
 of the oh pathway along with the mlliken 
 spin populations on selected atoms for each of the critical points 
 on the path ( see si figure s3 for the energy 
 profile for the h2o pathway ) .
relative energies ( in kcal / mol , with fe  of as the reference state ) of reactant , ts and 
 i1 and mlliken spin populations of key atoms ( fe , the first 
 o denotes the oxo , the second o denotes the o of oh bound to the iron , and c denotes the carbon of methyl group of 1-mea ) 
 for the hydrogen abstraction step for oh pathway 
 in quintet ( fe  of and fe  oaf ) and triplet states 
 ( fe  of and fe  oaf ) . similar to the h2o pathway , the lowest energy 
 state 
 in the hydroxyl pathway for the reactant r is the fe  of substate . in the same 
 manner , for the transition state ( ts )
tsri1 and intermediate i1 structures , the fe  oaf substate has the lowest energy .
as expected for the fe  of reactant , the distance between oh and fe ( 1.78 
  ) for the oh pathway is shorter than the 
 distance between h2o and fe ( 2.09  ) for the h2o pathway , and it is a sign of a stronger interaction between 
 oh and fe . as mentioned in section 2 , we replaced the h2o bound to the iron with oh without running md simulations for the oh pathway .
the total electrostatic potential ( esp ) fitted charge of 
 the hydroxyl is 0.3466 ( sum of the h and o charges ) in the 
 optimized reactant , which means part of the negative charge on oh is delocalized because of its coordination to the 
 iron .
it is possible that the oh may have an impact 
 on its surrounding mm environment due to the change in charge , although 
 this effect might not be significant due to the charge delocalization . 

the 
 calculated energy barrier for the oh pathway 
 ( 23.2 kcal / mol ) is 0.8 kcal / mol higher than the barrier calculated 
 for the h2o pathway ( 22.4 kcal / mol ) .
the slight energy difference indicates that the h2o and oh pathways are almost equally preferred 
 for the hydrogen abstraction step .
the intermediate i1 is 1.4 kcal / mol higher than the reactant r for the 
 oh pathway , compared to 3.7 kcal / mol lower than 
 r for the h2o pathway ( figure s3 ) . 

this result suggests oh stabilizes the reactant 
 more than the intermediate , whereas in the presence of water the opposite 
 is observed . in the present studies , we have not performed free energy 
 calculations based on the minimum energy paths ( meps ) , since the potential 
 energy barrier for the rate limiting step for the
thus , we 
 expect the associated free energies to show a similar trend as observed 
 before .
moreover , in our previous study , 
 it was found that the mm environment has only a minor impact on the 
 free energy barrier for the rate limiting step , and it appears its 
 major role for this step is to maintain the geometry around the fe 
 and its ligands . despite the slight difference in the 
 barrier , the electronic structure of the fe 
oxo 
 moiety exhibits different features for the oh pathway 
 because of the equatorial oh ligand . for the h2o pathway , the number of unpaired electrons of the iron and 
 oxo o atoms increases during the elongation of the fe 
o distance 
 ( d(fe  ooxo ) ) for both fe  of and fe  oaf ( see si table s1 ) .
for the oh pathway , the trend is the same for fe  oaf ( see table 1 ) .
however , for fe  of , the elongation of d(fe  ooxo ) causes an increase in the number of unpaired electrons on the iron 
 and a decrease on the oxo , which suggests one electron is transferred 
 back to the oxo from the iron .
note that we modified the d(fe  ooxo ) by only moving the oxo and fixing all 
 other atoms from fe  oaf reactant instead of relaxing the whole complex with frozen d(fe 
the numbers 
 in italics correspond 
 to the structure where the fe  oaf and fe  of states 
 are close in energy ( toh ) . except for this point ,
all other 
 structures are obtained by only moving o with all other atoms fixed 
 starting from the optimized fe 
oaf reactant . to understand 
 these differences , we turn to the analysis of the 
 canonical molecular orbitals for both pathways . during the h atom 
 abstraction
, an electron will be transferred to the oxo o. figure 3 shows the -lumo ( lowest unoccupied molecular 
 orbital ) and -lumo for the reactant structures for the h2o pathway .
for the fe  of , the -lumo (  * orbital ) only changes slightly 
 during the elongation of the d(fe  oxo ) from 
 the reactant ( 1.60  ) to the mecp ( 1.78  ) .
in contrast , 
 the percentage of o in -lumo (  * orbital ) increases and 
 starts to dominate .
-lumo and -lumo 
 ( canonical orbitals , isovalue = 0.05 
 au ) of the quintet reactants , mecp and ts structures along the h2o pathway .
carbon atoms are colored in gray , hydrogen in white , 
 nitrogen in blue , oxygen in red , iron in purple , and boundary carbon 
 atoms for pseudo - bond in cyan . according to previous studies on the reactivity of fe  oxo ,
the path for an 
  electron being transferred from the substrate to the -lumo 
 has been referred to as the  channel , and the path for a  
 electron being transferred from the substrate to the -lumo 
 has been proposed as the  channel . for the  channel ,
the 
 substrate approaches the fe  oxo in a colinear 
 fashion from the top to maximize the overlap between their orbitals . 
 for the  channel , the substrate
however , the pauli repulsion 
 between them will make the angle close to 120 instead of 90. 
 the two channels were proposed to arise from the same fe  of reactant and form a fe  oxyl radical on the way to the ts . as shown in figure 3 , the fe  oaf and fe  of transition state ( ts ) structures for the h2o pathway correspond to the tss for the previously proposed 
  and  channels respectively originating from the same fe  of reactant .
however , the fe  oaf state , which may become the ground state at a relatively long d(fe 
ooxo ) was not considered in previous 
 studies as it might be in a relatively high energy level for the complexes . 

the -lumos of both reactant and mecp in fe  oaf are mostly comprised of oxygen s 
 p orbital with little covalency with the iron s d orbital ( see 
 figure 3 ) .
therefore , the moiety in fe  oaf can be characterized as an 
 oxyl weakly coupled with fe(iii ) , which is a good  electron 
 acceptor from the substrate . moreover ,
the fe  oaf is more stable than fe  of at a relatively long d(fe  oxo ) 
 when approaching the ts .
however , one disadvantage for fe  oaf is that its -lumo is 
 perpendicular to the iron 
similar to 
 the  channel mentioned above , the substrate tends to approach 
 the iron 
oxo horizontally to maximize the orbital overlap but 
 causes large pauli repulsion , this interplay yields a bent fe  o  h 
 angle . for the model complexes used in previous studies , where the 
 substrate can move freely
, both channels can be accessed from the same state . 
 in these studies ,
the fe  of ( called fe(iv)oxo in these studies ) is the lowest energy 
 state .
thus , the  channel for fe  of , which involves less pauli repulsion , is a 
 better choice in that case than the fe  oaf state .
considering all the factors discussed above , 
 the  channel 
 starting from the fe 
oaf reactant may compete with the  channel arising from the fe  of reactant to accept 
 the  electron from the substrate in the enzyme environment . 

oaf state being stabilized by surrounding ligands , 
 or the substrate being oriented in a non - colinear arrangement to fe  o 
 due to steric constraints . to access the  channel ,
the complex 
 needs to transit from fe  of to fe  oaf . for the 
 h2o pathway
, the crossing between these two states happens 
 via a minimum energy crossing point ( mecp ) .
the angles formed by the fe  o  h atoms for these two 
 quintet states at the ts are around 130 , and the d(fe  oxo ) for the fe  oaf ( 1.80  ) reactant is close to the one for the lowest 
 ts ( fe  oaf , 1.77  ) 
 and larger than the value for the fe  of reactant ( 1.60  ) .
it can 
 be seen from figure 3 that the lumos of fe  oaf ts resemble those 
 of fe  oaf mecp and the 
 lumos of fe  of ts resemble 
 those of fe  of mecp . 
 the transition from the fe  of reactant to a low - lying state at a relatively long d(fe 
o ) , the description of which matches the features 
 of fe  oaf , also occurs 
 and results in the non - colinear fe  o  c in the fe 
oaf ts for some simple model complexes 
 with free substrates and small - size negative charged ligands such 
 as [ fe(o)(f)5 ] .
regarding the two quintet reactants 
 for the oh pathway ( see figure 4 for their lumos ) , the -lumo of the fe  of reactant contains a large component 
 from 1-mea , and the orbital of fe  oxo is not in a good orientation 
 for overlapping with the orbitals of the methyl group of 1-mea . in 
 contrast , the -lumo of fe  oaf is still mainly comprised of oxygen s p orbital and 
 should be more likely to accept an  electron from the substrate . 

it was expected that the elongation of d(fe  o ) 
 would lead to more electrons being transferred from the oxo to the 
 iron for both substates and complete the state transition via a mecp 
 like the h2o pathway .
however , the change of mlliken 
 spin population in table 1 shows the elongation 
 of d(fe  oxo ) leads to a  electron being 
 transferred from the iron to the oxo for fe  of , and smoothly change into fe  oaf . in practice , the optimization after 
 the structure ( toh , in table 1d(fe  oxo )
= 1.79  ) that has a small energy 
 difference for fe  oaf ( and fe  of ) gives the 
 result that both states converge to fe  oaf . in terms of d(fe 
o ) , the value 
 is 1.62  in fe  of reactant and 1.88  in fe  oaf reactant and ts , which suggests that the fe 
-lumo and -lumo 
 ( canonical orbitals , isovalue for 
 the surface is 0.05 au ) of the quintet reactants , toh ( table 1 ) and ts structures along the oh pathway .
carbon atoms are colored in gray , hydrogen in white , nitrogen 
 in blue , oxygen in red , iron in purple , and boundary carbon atoms 
 for pseudo - bond in cyan .
figure 4 shows the lumos (  
 and  ) 
 of the fe  oaf ts also 
 resemble the respective lumos of the fe  oaf reactant .
in addition , different from the fe  of reactant where its 
 -lumo is mainly comprised of the orbitals for the substrate , 
 the -lumo in fe  of toh is mainly a fe  o  * orbital , which is 
 more similar to the -lumo of the fe  of ts .
oxo(oxyl ) distance , d(fe  ooxo ) , in tuning the reactivity of the fe 
in order to obtain the precursor for the fe  of ts , the oh also 
 needs to be considered to investigate the electronic structure change 
 from the fe  of reactant 
 to the fe  of ts . since 
 this pathway is on a higher energy level than the mep ( minimum energy 
 pathway ) , it will be not discussed here . when studying the hydrogen 
 abstraction by fe 
oxo , the fe  oaf state should be paid attention , as 
 it may become the ground state on the way to the ts , especially for 
 equatorial electron donating ligands and/or enzymes with constrained 
 substrates .
the lowest energy states for i1 and i1 are both fe  oaf , and the two substates 
 for the quintet and triplet merge into one after the oh rebound step 
 at i2 and i2 .
therefore , only the quintet state is considered 
 for the calculations of this and subsequent steps .
the calculated 
 barrier , tsi1i2 , is 12.2 kcal / mol , which is in agreement 
 with a previous qm / mm study .
for the oh pathway , the corresponding intermediate i3 is not stable . during the optimization ,
the proton is transferred 
 to the iron - bound oh spontaneously , and the zwitterion 
 structure i2 forms .
relative energies ( kcal / mol ) of the structures 
 along the minimum 
 energy path ( mep ) for the oh rebound step in the quintet state for 
 h2o pathway ( a ) and oh pathway ( b ) . 

the numbers in the parentheses are reaction barriers , which are the 
 energy differences between intermediates and their corresponding tss . 

the energy of the corresponding fe  of reactant ( figure 2 ) is taken as zero 
 for each pathway .
carbon atoms are colored in gray , hydrogen in white , 
 nitrogen in blue , oxygen in red , iron in purple , and boundary carbon 
 atoms for pseudo - bond in cyan .
one is a concerted pathway in which the oh rebound process 
 is coupled to the proton transfer via tsi1i2 .
the other is a stepwise pathway where the proton is first transferred 
 to form an intermediate i4 , followed by an oxygen transfer . 

the calculated tsi1i2 for the concerted pathway 
 is 11.9 kcal / mol . for the stepwise pathway ,
the energy for the tsi1i4 structure between i1 and i4 is 24.7 kcal / mol , and the subsequent oxygen transfer is 
 a barrierless downhill process .
based on these barriers , the concerted 
 pathway is favored over the stepwise pathway . regarding the zwitterion 
 structure i2 ,
the distance between fe and o of ch2o ( on the adenine base ) is short ( 2.05 
  ) .
as shown in figure 6 , among the ligands 
 coordinated to the iron , the nci surface between the ch2o moiety and the fe atom has the deepest 
 blue color .
the nci result suggests that the zwitterion structure also 
 exhibits the characteristic fe  o  c bond proposed for 
 the taud mechanism from time - resolved raman studies .
similarly , one may expect a concerted pathway to be preferred 
 over a stepwise pathway as proposed for taud from the radical intermediate 
 to the fe  o  c intermediate .
nci 
 surface ( isovalue 0.5 au and a color scale 0.1 < 
 sign(2) < 0.1 au ) of the zwitterion structure 
 ( i2 ) for the oh pathway . the lowest barrier for the oh pathway ( tsi1i2 , 12.0 kcal / mol ) is close 
 to the one for the 
 h2o pathway ( tsi1i2 , 12.2 kcal / mol ) .
the minor 
 difference indicates these pathways are also equally favored for this 
 step . for both pathways
, the rebound step leads to an intermediate 
 with much lower energy with respect to the fe 
whether a zwitterion structure denoted as 
 i5 also exists for the h2o pathway , we carried out the 
 optimization starting from a structure in which the proton is transferred 
 from ch3oh to asp133 . during the optimization
, 
 the proton is spontaneously transferred back to ch3o , which suggests that the pka of the iron - bound asp133h is larger than 
 the iron - bound ch3oh when a hydroxyl is coordinated 
 to the fe atom . in order to investigate the effect from the 
 positive charge on 
 the nitrogen ( n1 ) of 1-mea , we changed 1-mea to 1-deazamea by replacing 
 n1 with c ( see figure 7a ) in the optimized 
 zwitterion i2 ( bound to the iron ) and i5 ( unbound from the iron ) structures and carried out geometry optimizations . 

a stable structure for the i2 analog ( figure 7b ) for 1-deazamea was obtained on the pes .
conversely , 
 in the case of the i5 analog for 1-deazamea , the ch2o group abstracts a proton spontaneously 
 from the neighboring water that is coordinated to the iron ( figure 7c ) .
this finding is consistent with previous results 
 for 3-deazamec . additionally , different 
 from 1-mea
, we are able to obtain the i3 analog ( figure 7d ) for 1-deazmaea , although its energy is 8.0 kcal / mol 
 higher than for the i2 analog .
these optimized structures 
 indicate that the proton accepting ability of iron - bound ch2o group in 1-deazamea is weaker than when 
 this group is not bound to the iron because of the stabilization effects 
 from the metal .
the pka of iron - unbound 
 ch2oh in 1-deazamea is larger than the value of 
 h2o coordinated to the iron , and hence larger than 1mea . 

since the proton transfer is a necessary step for the repair process 
 and might become the rate - limiting step as discussed in the next section , 
 the pka difference of ch2oh may partly explain why 1-mea and 3-mec , which bear a positive 
 charge , are preferred by alkb over 3-met and 1-meg , which are charge 
 neutral alkylated dna bases .
carbon atoms are colored in gray , hydrogen in white , 
 nitrogen in blue , oxygen in red , iron in purple , and boundary carbon 
 atoms for pseudo - bond in cyan . to study
whether the unbinding of the methoxide moiety from the 
 metal ( figure 8 ; from i2 to i3 for the h2o pathway ; from i2 to i5 for the 
 oh pathway ) is a necessary step , we investigated 
 the formation of the formaldehyde with ch3oh ( for 
 the h2o pathway ) or ch3o ( for the oh pathway ) being coordinated or unbound 
 to the iron . in the case of the h2o pathway with iron - coordinated 
 ch3oh
, the reaction happens in a concerted manner , 
 where the proton transfer to asp133 and the bond breaking between 
 the c and n1 of 1-mea ( c  n bond breaking , i2p pathway 
 in scheme 2 ) leads to the product with the 
 formaldehyde bound to the iron ( p ) .
the calculated barrier for this 
 step is 25.9 kcal / mol , which is higher than the hydrogen abstraction 
 step .
this result suggests that this step may become the rate - determining 
 step under certain circumstances , such as if no better proton acceptors 
 are available .
relative energies ( in kcal / mol ) for the structures along 
 the minimum 
 energy path ( mep ) for the detachment of the dna base from fe and the 
 formation of formaldehyde in the quintet state for the h2o pathway ( a ) and oh pathway ( b ) .
the numbers 
 in the parentheses are reaction barriers , which are the energy differences 
 between intermediates and their corresponding tss .
the fe  of reactant ( figure 2 ) is taken as the reference for each pathway .
carbon atoms 
 are colored in gray , hydrogen in white , nitrogen in blue , oxygen in 
 red , iron in purple , and boundary carbon atoms for pseudo - bond in 
 cyan .
when the ch3oh moiety is leaving the iron , i3 
 forms , and its energy is 5.2 kcal / mol higher than the iron - coordinated 
 intermediate i2 .
n bond breaking . 
 if these two processes are stepwise , a zwitterion intermediate ( i4 ) 
 that is unbound to the iron will form .
the proton can then be transferred 
 to asp133 or some other neighboring residue , such as glu136 , in a 
 direct - transfer pattern or via a water bridge . however , in the case 
 of asp133 , the proposed zwitterion structure can not be obtained , which 
 is partly due to its weaker ability to accept a proton than for iron - unbound 
 asp133 . in other words ,
the proton transfer to asp133 has to be coupled 
 with the c  n bonding and form the final product with the formaldehyde 
 unbound to the iron ( pun ) .
the barrier ( tsi3p ) for this coupled process is 20.6 kcal / mol , and its relative energy 
 is close to the unbound tsi2p .
it is also possible 
 that the proton is transferred to a neighboring 
 residue instead of being transferred to asp133 . as shown in figure 1 , the nearest residue to the active site
is glu136 , 
 but its distance from the 1-mea suggests the proton transfer would 
 likely have to occur via a water bridge . before the proton transfer , 
 the structure rearranges from i3glu136 to i3glu136 .
the ch3oh moiety forms a hydrogen bond with 
 asp133 while ch3oh forms a hydrogen bond with the 
 bridging water in i3glu136 . to check the existence 
 of a zwitterion structure , we carried out the optimization of i4glu136 assuming the proton transfer from ch3oh to glu136 with a water molecule as the bridge .
however , during 
 the optimization , the structure changes back to i3glu136 with the proton being spontaneously transferred back .
this indicates 
 that the pka of ch3oh is also larger than glu136h . in other words , if the proton is 
 transferred to glu136 , it has to be coupled with the c 
the calculated barrier for tsi3pun - glu136 for the concerted pathway from i3glu136 to pglu136 is 7.4 kcal / mol , which is much lower than the barrier 
 for the proton being transferred to asp133 .
this suggests that glu136 
 may be the final proton acceptor when no better acceptor available . 

the proton acceptor role of glu136 may partly account for the decreased 
 activity of alkb in repairing 1-mea when glu136 is mutated to a leucine .
relative energies ( kcal / mol ) for the structures along 
 the minimum 
 energy path ( mep ) for the proton transferred to glu136 in the quintet 
 state for the h2o pathway .
( arg210 , glu136 , and a bridging 
 water were added to the qm subsystem for these structures ) .
carbon 
 atoms are colored in gray , hydrogen in white , nitrogen in blue , oxygen 
 in red , iron in purple , and boundary carbon atoms for pseudo - bond 
 in cyan .
the structure of the tsi3pglu136 shows a proton 
 is first transferred from h2o to glu136 , followed by the 
 resulting oh accepting the proton from ch3oh .
it is worth noting that asp135 could be another possible 
 proton acceptor as well .
however , as its relative position to the 
 dna base in the crystal structure ( figure 1 ) is not conducive for the proton transfer , the proton transfer process 
 may happen when the dna base is leaving the active site .
if a zwitterion 
 structure ( i4 ) indeed forms , the proton from ch3oh could be transferred to a hydroxyl molecule in the solvent . in 
 summary , for the h2o pathway , after the hydrogen abstraction 
 and the oh rebound step , the hydroxyl product first unbinds from the 
 iron and loses a proton to glu136 or asp135 or solvent with concerted 
 the c 
for the oh pathway , 
 as the proton has already 
 been transferred to the iron - bound oh in the previous 
 rebound step , the final step is only the c 
the calculated barrier when the methoxide is bound to the 
 iron ( tsi2p ) is 5.9 kcal / mol , compared to 
 the unbound structure ( tsi2i5 ) that results 
 in a barrier of 17.3 kcal / mol .
this last barrier is 3.0 kcal / mol higher 
 than the barrier for the unbinding process for ch3oh , which may be due to a stronger attraction between the iron and 
 o of ch3o than ch3oh and the repulsion between the negatively charged o of ch3o and one o of asp133 during the unbinding 
 process .
the iron - unbound zwitterion intermediate i5 is 
 only 0.6 kcal / mol higher than the iron - bound one ( i2 ) . 

the barrier for i5 being finally dissociated into pun ( tsi5p ) is 1.9 kcal / mol . 

the imaginary frequency vibrational mode corresponds to the rotation 
 of the formed formaldehyde , which suggests the energy for c 
n 
 bond breaking should be lower than 1.9 kcal / mol . therefore , for the 
 oh pathway , after the hydrogen abstraction and 
 the oh rebound coupled with a proton transfer to the oh , the formed zwitterion structure prefers the c 
for both the h2o and oh meps ( see si figures 
 s4 and s5 for the complete energy profile for these two pathways 
 respectively ) , the rate - limiting step is the hydrogen abstraction 
 step , and their barriers for this step are close to each other .
once 
 the rate limiting step has been achieved , the mechanisms differ significantly . 
 for the h2o pathway ,
the last step with the lowest barrier 
 is a proton transfer to glu136 via a water bridge , and the hydroxyl 
 product has to unbind from the iron center first . since the water 
 molecule that acts as the bridge and glu136 can move freely , they 
 may not always be in a perfect arrangement for the proton transfer . 
 in that case
, asp133 may become the best choice . as the barriers for 
 a proton being transferred to asp133 coupled with the c 
n bond 
 breaking are higher than the hydrogen abstraction step , this step 
 may become the rate - limiting step for the h2o pathway . 

in contrast , the barrier for the last step for the oh pathway , the c 
n bond breaking leading to the formation of 
 formaldehyde is much lower . taken together
, our results suggest that 
 the oh pathway should be preferred over the h2o pathway , which may partly account for the basic optimal 
 ph for the 1-mea repair catalyzed by alkb .
the crystal structure 
 for 3-mec ( 3ois , si figure s1 ) is proposed to be a zwitterion 
 structure similar to i4 where the base is unbound to the metal based 
 on qm / mm calculations .
however , those 
 qm / mm calculations can not rule out the possibility of an alcohol structure . 
 according to our results on 1-mea above , the zwitterion structure 
 generated by just following the
oh pathway is more 
 likely to dissociate with the methoxide moiety bound to the metal , 
 rather than unbinding from the metal .
in addition , if the captured 
 crystal structure for 3-mec ( 3ois ) is indeed a zwitterion , the proton 
 from the alcohol structure has to be transferred to the solvent instead 
 of to asp133 or glu136 . as a result
, another possible pathway may 
 be proposed where the hydrogen abstraction , oh rebound , and unbinding 
 from the iron follow the h2o pathway , and then , the proton 
 is transferred to oh to form a zwitterion structure . 

in this 
 work , new pathways for the second part of the reaction 
 mechanism , starting from the rate - limiting h atom abstraction , for 
 the dealkylation of 1-mea catalyzed by alkb have been proposed and 
 investigated by qm / mm simulations based on recent experimental findings . 
 for the hydrogen abstraction and the oh rebound step , the h2o and oh pathways have close barriers and
therefore 
 are equally preferred . for the hydrogen abstraction step , different 
 from most of the previous studies on model systems , the fe  oaf state ,
where the iron ( s = 
 5/2 ) is antiferromagetically coupled with a oxyl ( s = 1/2 ) 
 becomes the ground state when the fe  oxo distance , d(fe  ooxo ) , is long and similar to the fe 
this finding highlights 
 the electronic 
 structure change of the fe  oxo moiety with the 
 binding of the equatorial oh under the enzymatic 
 environment . regarding the oh rebound step , a hydroxyl structure forms 
 for the h2o pathway .
in contrast , for the oh pathway , this step is coupled with the proton transfer from ch3oh to the oh bound to the iron and forms 
 a zwitterion structure bound to the iron , which can be also characterized 
 as an fe  o  c complex , which is consistent with experimental 
 findings .
n bond between 
 ch3o and the dna base can easily 
 break to form the final product while the dna base is bound to fe . 

in contrast , for the h2o pathway , the hydroxyl complex 
 needs to unbind from the iron center first and then transfer a proton 
 to the neighboring residue glu136 via a water bridge or asp135 or 
 lose it to the solvent . the proton transfer to glu136
. the larger pka value of ch3oh in the hydroxyl intermediate for 
 neutral dna bases may account for its lower repair efficiency compared 
 to positively charged dna bases .
the lower energy barrier for the 
 last step in the oh pathway compared to that of 
 the h2o pathway when the proton has to be transferred to 
 asp133 may partly explain the basic optimal ph for the repair of 1-mea 
 by alkb .
comparison of the energetics for the oh rebound step of 1-mea 
 and 1-deazamea show that the positive charge on the n 
 of 1mea is necessary to reduce the barrier , and may help explain alkb s 
 substrate preference .
 alkb 
 is the title enzyme of a family of dna dealkylases that catalyze 
 the direct oxidative dealkylation of nucleobases . the conventional 
 mechanism for the dealkylation of n1-methyl adenine ( 1-mea ) 
 catalyzed by alkb after the formation of feiv  
 oxo 
 is comprised by a reorientation of the oxo moiety , hydrogen abstraction , 
 oh rebound from the fe atom to the methyl adduct , and the dissociation 
 of the resulting methoxide to obtain the repaired adenine base and 
 formaldehyde . 
 an alternative pathway with hydroxide as a ligand bound 
 to the iron atom is proposed and investigated by qm / mm simulations . 
 
 the results show oh has a small impact on the barriers 
 for the hydrogen abstraction and oh rebound steps . 
 the effects of 
 the enzyme and the oh ligand on the hydrogen abstraction 
 by the feiv  
 oxo moiety are discussed in detail . 
 
 the new oh rebound step is coupled with a proton transfer to the oh ligand and results in a novel zwitterion intermediate . 
 
 this zwitterion structure can also be characterized as fe  o  c 
 complex and facilitates the formation of formaldehyde . 
 in contrast , 
 for the pathway with h2o bound to iron , the hydroxyl product 
 of the oh rebound step first needs to unbind from the metal center 
 before transferring a proton to glu136 or other residue / substrate . 
 
 the consistency between our theoretical results and experimental findings 
 is discussed . 
 this study provides new insights into the oxidative 
 repair mechanism of dna repair by nonheme feii and -ketoglutarate 
 ( -kg ) dependent dioxygenases and a possible explanation for 
 the substrate preference of alkb . 
Introduction
Computational Methods
Results and Discussion
Conclusions