ribonucleotide reductases 
 ( rnrs ) catalyze the conversion of four 
 nucleotides ( cdp , udp , adp , gdp ; ndps ) into the corresponding deoxynucleotides 
 ( dndps ) .
class ia rnrs are found in nearly all eukaryotic 
 and some prokaryotic organisms and are 
 composed of two homodimeric subunits , 2 and 2 , which 
 form an active , transient 22 complex .
subunit 2 houses the catalytic substrate binding 
 site and the binding sites for the allosteric effectors that govern 
 specificity and activity of nucleotide diphosphate ( ndp ) reduction .
subunit 2 houses the diferric - tyrosyl radical cofactor ( the 
 fe2-y ) essential for initiating ndp 
 reduction . during each turnover , the fe2-y-2 
 oxidizes c439 in the active site of 2 where dndp 
 is produced , and then it is subsequently reoxidized .
the oxidation 
 occurs via a radical hopping mechanism over 35  between the 
 two subunits along a specific pathway comprised of redox active amino 
 acids ( y122  [ w48 ? ]  y356 in 2  y731  y730  
 c439 in 2 ) .
reversible oxidation and reduction of 
 y during turnover requires release of the phenolic proton to an acceptor 
 concomitant with the oxidation ( figure 1a ) , 
 a mechanistic strategy to avoid formation of high - energy intermediates .
while the long - range oxidation through aromatic 
 amino acid residues in ribonucleotide reductase ( rnr ) is unprecedented 
 in biology , proton - coupled 
 electron transfer ( pcet ) mechanisms are involved in many fundamental 
 processes in biology including photosynthesis , respiration and nitrogen 
 fixation .
rnrs can thus serve as a paradigm for understanding 
 pcet in a complex biological machine and , in comparison with other 
 systems , offer the opportunity to identify common principles that 
 control this basic transformation .
since pcet is intrinsically of 
 quantum mechanical nature because of both electron and proton tunnels , 
 the large difference in mass ( factor 2000 ) causes the proton 
 translocation to be limited to very short distances ( < 1  ) , while the electron may transfer over very long 
 distances .
thus , in pcet the electron and the proton might be transferred 
 to different acceptors ( orthogonal or bidirectional ) or they might 
 move between the same donor / acceptor pairs ( colinear ) .
description 
 of the coordination of these events has been theoretically challenging 
 and is summarized in recent reviews .
investigation 
 of the electron and proton pathways has required rnrs with site - specifically 
 incorporated unnatural amino acids and electron paramagnetic resonance 
 ( epr ) combined with electron  nuclear double resonance ( endor ) 

spectroscopies have provided a unique opportunity to access mechanistic 
 details if the intermediate radicals can be detected . the current 
 model for this long - range oxidation
it was originally proposed based on the x - ray 
 structures of 2 and 2 , a docking model of these subunits 
 based on their shape complementarity , and conserved residues .
recent biophysical studies including pulsed electron  electron 
 double resonance ( peldor ) studies and small - angle 
 x
- ray scattering studies ( saxs ) and cryoem have established that the docking model is a 
 reasonable representation of the  active  rnr structure 
 in solution and that the oxidation occurs over > 35  .
unfortunately 
 there is little information about the molecular details of the interface 
 between  and  , more specifically , the communication 
 between y356 in 2 and y731 in 2 . 

the last 2530 amino acids of all 2 structures which 
 include y356 or its equivalent are structurally disordered 
 and the last 15 amino acids of escherichia 
 coli 2 are largely responsible for 
 the weak interaction between the subunits ( kd of 0.1 
 to 0.2 m ) . working model for pcet in e. coli rnr .
( a ) scheme 
 for concerted pcet between pathway tyrosines within . ( b ) amino 
 acids as well as protons ( blue ) proposed to be involved in radical 
 transfer between the  and  subunits . w48 and 
 d237
are shown in gray because there is currently no evidence 
 for their participation in pcet . in  ,
the protons are proposed 
 to move orthogonal to the electrons ( blue vs black arrows ) and within 
  they are proposed to move colinear with the electrons ( purple 
 arrows ) .
( c ) overlay of residues in  from the resting state 
 x - ray structure ( pdb 2x0x gold / green ) with the density functional 
 theory ( dft ) optimized structure for radical state y730 ( residues y731 , y730 and c439 in gray shade ) .
position of 
 the conserved water molecule wat1 as well as on - pathway ( yellow dots ) 
 and other ( red dots ) hydrogen bonds are from the dft structure .
the 
 h bond distance of y731 to y730 is ro730o731  2.6  whereas 
 the h bond distances of c439 and wat1 to y730 are rs439o730  
 3.3 and rowat1o730  2.8 
  respectively .
inset : 
 chemical structure of nh2y and ys in the pathway 
 replaced by nh2y as well as the double mutants employed 
 in this study .
studies of this unprecedented 
 oxidation catalyzed by wt rnr have 
 not been possible as protein conformational change(s ) constitute the 
 rate - limiting step(s ) and kinetically mask all of 
 the detailed chemistry of the radical transfer ( rt ) and the nucleotide 
 reduction step . however , use of technology 
 in which the pathway tyrosines are replaced site - specifically with 
 tyrosine analogues with altered pkas and reduction potentials , and the development of high frequency ( hf ) epr and endor methods to characterize y 
 analogues generated with the mutant rnrs , are now unmasking the chemistry 
 of this long - range oxidation .
these studies taken together suggest 
 that conformational gating is occurring within  and that radical 
 intermediates formed at y356 , y731 , y730 , c439 and substrate radical are likely in equilibrium 
 along the pathway .
their relative redox potentials 
 are progressively uphill with corresponding lower concentrations . 
 however , there is sufficient concentration of the most uphill radical 
 ( substrate radical ) to drive the reaction to the right by a rapid , 
 irreversible step proposed to be the loss of water during nucleotide 
 reduction . once the chemical reaction is complete , the reformation 
 of the y122 in  is energetically downhill .
the results from the incorporation of nh2y site - specifically 
 at 356- , 731- and 730- , the focus of this paper , have 
 played a critical role in our current understanding of specific steps 
 of the rt pathway and the conformational gating process . in all three 
 cases , nh2y-( )
is generated in 3040% 
 yield upon incubation of the second subunit ( ) , substrate 
 cdp and allosteric effector atp .
the formation of the nh2y is a 
 biphasic process with both phases kinetically competent in nucleotide 
 reduction at 5 to 10% the rate of the wt - rnr .
three recent experiments using this probe address the importance 
 of pcet and the 22 conformational changes triggered 
 by binding substrate and effector .
insight into the initial rt event 
 within  has been studied by rapid freeze quench ( rfq ) mssbauer 
 analysis using wt-2/nh2y730-2/cdp / atp .
the results established that the proton of the 
 water bound to fe1 ( figure 1b ) is transferred 
 to y122 coupled with an electron , likely from y356 . using the nh2y730-2 only 
 the forward radical step is observed and interestingly the fe1-oh 
 now remains .
these results support the model of orthogonal pcet in 
 2 and the exquisite control dependent on the proton s 
 location ( figure 1b ) . using the same 
 experimental design , analysis of the nh2y by hf 
 epr and deuterium ( h ) endor provided 
 the first spectroscopic insight suggesting the importance of colinear 
 pcet within 2 and the structural relationship of y731 and y730 in the active 22 rnr complex . the link between epr data in the active intermediate state and the x - ray structural data in the inactive 
 state
was supported by quantum chemical calculations of the energy 
 optimized structural models of y730- and 
 nh2y730- including 211atoms ( figure 1c ) .
the endor analysis in conjunction with density 
 functional theory ( dft ) calculations revealed one strong ( ro  o  2.7  ) and one weaker 
 ( rs  o  3.4  ) h bond 
 proposed to be associated with residues adjacent to nh2y730 in the pathway , as well as another weak ( ro  o  3.0  ) interaction 
 with water .
the water molecule was proposed to tune the rt rates in 
 wt enzyme around y730 by about 1 order of magnitude .
finally , our recent saxs and pull - down experiments 
 of rnr from this reaction mixture established that a single h atom 
 transfer from nh2y730 to y122 
 is sufficient to increase the weak / subunit affinity 
 by a factor of 10 .
this result 
 again highlights the importance and subtlety of conformational changes 
 in the rt process . in the current paper , experiments
in which 
 nh2y has 
 been site specifically incorporated at nh2y356- , and at nh2y731 and nh2y730- ( figure 1c ) in conjunction 
 with hf epr and hf h endor spectroscopies , density functional 
 theory ( dft ) calculations using nh2y731(730)- structures , have been employed in an effort to understand 
 the electrostatic and h bonding environment at these residues in the 
 rt pathway .
hf epr data were recorded at a microwave frequency of 
 263 ghz , which delivers unprecedented resolution in the spectra of 
 the nh2ys .
in addition , double mutants nh2y730/c439a- and nh2y731/y730f- were prepared and the h endor results compared to the single mutant results in an effort 
 to assign the source of the h bonds observed to specific residues . 

our results support the colinear concerted pcet model within  
 and provide spectroscopic evidence for the unusual stacking of y730/y731 in the active 22 complex .
to probe the electrostatic environment of the 
 radical intermediates at the three different positions in the rt pathway 
 we examined the hf epr spectra formed after incubation of -nh2y356 , -nh2y731 , -nh2y730 with their respective complementary wt subunits , 
 either  or  , cdp , and atp ( methods ) .
figure 2 presents 263-ghz pulse epr spectra 
 from the individually trapped nh2ys recorded with 
 a prototype quasi - optical epr spectrometer ( methods ) .
the buffer was exchanged with d2o to enhance epr resolution . 
 at this frequency , all gx values of nh2ys ( nd2ys ) are 
 clearly resolved and shifted by about 1 ppt ( or 0.001 ) from the value 
 calculated for a free nh2y ( gx , free = 2.0061 ) .
this shift is significant , 
 on the order of the effect predicted from several hydrogen bonds.g values were 
 calibrated with the spectrum of the stable radical y122 in 2 , which is present in all samples and visible 
 only at low temperatures
. additionally , gx slightly decreases starting from the most buried 
 intermediate nh2y730 ( gx = 2.0054 ) to nh2y356 ( gx = 2.0049 ) 
 located at the subunit interface .
as a control , spectra were also 
 recorded in protonated buffer and the g values were best reproduced 
 ( supporting information , figure s1 ) .
simulations 
 of the 263 ghz spectra combined with 94 ghz spectra ( the latter frequency 
 was used to constrain the simulation , figure s2 ) led to a consistent set of g values and c- 
 proton hyperfine ( hf ) couplings ( see chemical structure in figure 1 inset ) that are summarized in table 1 . thus , the epr spectra of the single mutants in the active enzyme are contributed by a radical species ( per 
 mutant ) with a well - defined microenvironment ( g value ) 
 and molecular orientation .
this finding underlines the importance 
 of a specific structural arrangement in the active state to permit 
 radical propagation .
the observed shift of gx values is a hallmark for a substantial effect 
 of either positive charges and/or hydrogen bond interactions . since 
 hydrogen bonds are expected to have a predominant effect on g values , a possible correlation 
 of the observed gx shifts 
 with the number and strength of hydrogen bonds was examined .
. electron spin  echo ( ese ) detected spectra of 
 intermediates without ( top spectrum for nh2y731/y122 , 10 k ) and with relaxation filtering 
 ( bottom spectra for nh2ys , 70 k ) .
echo 
 sequence used to suppress the signal of y122 ( box ) 
 is based on differences in the transverse relaxation times ( t2 ) of the radicals : nh2ys 
 and y122. at t = 70 k , the signal 
 associated with the y122 decays during the acquisition 
 ( box , purple dotted line ) and does not contribute to the spin echo 
 signal .
ese detected epr spectra of the nh2ys radicals 
 in h / d exchanged buffer : nd2y730 ( black ) , 
 nd2y731 ( red ) and nd2y356 ( blue ) .
experimental conditions : ese ( /2echo ) 
 spectra : /2 = 60110 ns ,  = 290 ns , 250500 
 averages / point , acquisition time / spectrum = 1.53 days , t = 10 and 70 k. the values were 
 obtained from 
 combined simulations of the 263 and 94 ghz spectra and compared with 
 those obtained from dft calculations . the n hyperfine 
 tensor of the nh2y was not varied in the simulations 
 and kept ax = 2.4 mhz , ay = 1.6 - 5 mhz , az = 30.7 mhz .
uncertainty in g values is 
 about 0.05 ppt for the experiments and 0.5 ppt for dft calculations . 

we probed 
 exchangeable hydrogen bonds around the trapped nh2y 
 intermediates by endor spectroscopy at 94 ghz .
endor reveals the spectrum 
 of magnetic nuclei that are in the coordination sphere ( usually 5 
  ) of the observed radical .
after buffer exchange , exchangeable 
 protons are substituted by deuterons ( abbreviated d , with the nucleus 
 denoted h ) , which become visible by endor in the h resonance region .
figure 3a illustrates 
 the h mims endor spectra 
 of the three nh2y intermediates ( nd2y ) trapped under comparable conditions as in the epr experiments . 

the spectrum of nd2y730 was initially 
 reported in ref ( 25 ) , and it is included here for further analysis .
all three spectra 
 contain a broad , almost featureless background extending over 2 
 mhz , which arises from the strongly coupled amino deuterons .
additionally , 
 two samples , nd2y730 and nd2y731 , show pairs of sharp peaks in the region 
 1 mhz that is usually dominated by deuterons in the hydrogen 
 bond range ( figure 3a , peaks marked in red 
 and blue ) .
the spectra of nd2y730 and 
 nd2y731 look quite similar , with the 
 peaks from nd2y731 being slightly shifted 
 to larger couplings .
the sharp peaks are split by an additional small 
 coupling , i.e. , the quadrupol coupling .
the endor spectrum can also 
 detect weak couplings ( ro  h    2.1  ) if they can be resolved 
 from matrix deuterons ( matrix line ) .
indeed , additional small differences 
 are observed here also in the central resonance region ( 0.3 
 mhz ) . in nd2y730 , this region was proposed 
 to be associated with a water molecule ( wat1 , figure 1 ) conserved in the structure of wt 2 and nd2y730-2 .
interestingly 
 the spectrum of nd2y356 lacks any sharp 
 peaks and is associated with resonances of the amino deuteron and 
 a matrix line .
( a ) comparison between 
 spectra 
 of nd2y730 ( black ) , nd2y731 ( red ) , and nd2y356 ( blue ) .
simulation of the spectrum 
 for nd2y731 ( gray ) is representatively 
 decomposed into the contributions of the amino deuterons ( blue dashed 
 and dotted lines ) and of one hydrogen bond ( red dashed line ) .
spectrum 
 of nd2y730 is contributed 
 by one strong ( red peaks ) and one moderate ( blue peaks ) h bond .
( b ) spectra of double mutants nd2y730/c439a and nd2y731/y730f ( gray and red , 
 respectively ) .
simulation of the spectrum of the retained h bond ( red 
 peaks ) with parameters reported in table s2 . the contributions from hydrogen bonds , as assigned here , are illustrated 
 on the right .
parameters : mims endor with /2 = 20 ns , 
  = 200 ns , shot repetition time = 10 ms , random rf acquisition at 1 shot / point , acquisition time = 2455 
 h , t = 70 k. the same relaxation filtering mechanism 
 was used for the endor experiments at 70 k as for the hf epr spectra 
 ( cf .
endor spectrum is centered at the 
 larmor frequency 0 of h , i.e. , 21.9 
 mhz at a field of 3.3 t. further experimental evidence for the hydrogen bond at nh2y731 was derived from orientation selective hf h endor spectra recorded at different field positions in the 
 epr line .
figure 4a shows mims endor spectra 
 recorded at the canonical orientations b0  gx , gy and gz within the region of 1.5 mhz .
powder 
 patterns are still observed at orientations b0  gx and gz as orientation selection 
 is moderate for the large excitation bandwidth of the pulses (  
 1.8 mt ) as compared to the total epr line width (  8 mt ) ; however , 
 clear differences in the line shapes are visible .
the smallest hf 
 coupling , taken as the center of the sharp peak , is observed at b0  gz and the hf tensor displays a form |az|  |ay| > |ax| ( using the definition |ax| < |ay| < 
 |az| ) .
previous dft calculations 
 have indicated that such a tensor form is typical for a deuteron directed 
 almost perpendicular to the tyrosine ring plane , as previously reported 
 also for nd2y730 , and having the smallest component along the h bond .
such 
 a tensor reflects couplings that still contain some contribution from 
 a scalar interaction arising from orbital overlap . in the following
, 
 we define hydrogen bonds with these tensor properties as strong to moderate ( ro  h  1.72.0  ) .
the definition 
 used here is consistent with ref ( 10 ) but expanded by the definition of moderate bonds .
considering that the gx tensor component lies along the c  o bond and gy points to the side of the 
 amino group , the euler angles  , 
  ,  between hyperfine a and g tensor obtained from the simulation ( table 2 ) are consistent with a hydrogen bond directed almost perpendicular 
 to the plane of the nh2y731 .
simulations of the entire h 
 endor spectrum with the parameters obtained here additionally indicate 
 that the spectrum of a nd2y731 can be 
 well reproduced by the contribution of a single hydrogen bond ( figure 3a ) according to the observed intensity ratio of 
 the sharp peaks with respect to the amino deuteron resonances .
h 
 94-ghz mims endor spectra ( black lines ) were recorded at field positions 
 in the epr line parallel to the canonical orientations of the g tensor , i.e. , for b0  gx , gy , gz .
simulations of the spectra ( red dashed - dotted curves ) were performed 
 as described in the methods section .
= 320 ns , rf pulse length = 40 s , shot repetition time = 150 
 ms , acquisition time = 50 h / spectrum , random rf acquisition , t = 10 k. the low s / n required 
 operation at very low t and contribution of y122 could not be separated here .
however , 
 y122 does not display any hydrogen bonds 
 but only a matrix line , as also discussed in ref ( 25 ) .
the inset shows the orientation 
 of the h hf tensor from the simulation . in an effort to assign 
 the h bonding interactions 
 observed with nh2y731- and nh2y730-2 by h endor , double mutants
nh2y731/y730f and nh2y730/c439a were generated , in which one of the proposed h 
 bonds was removed .
the proteins were expressed , purified to homogeneity 
 and characterized by stopped flow - vis spectroscopy monitoring the 
 rate of loss of y122 ( 410 nm ) and rate of formation 
 of the nh2y ( 320 or 325 nm ) in the presence of 
 wt-2/cdp / atp , figure s3 .
the results 
 are summarized in table s1 and compared 
 with results from similar experiments on the single mutants . with nh2y731/y730f-2
, nh2y was formed with biphasic kinetics 
 resulting in 34  3% conversion , similar to the amount formed 
 with nh2y731-2 ( 32  3% ) .
the kinetics of both phases , however , were slower : 
 1.5  0.1 and 0.3  0.03 s compared 
 to 9.6  0.6 and 0.8  0.1 s. similar 
 studies with nh2y730/c439a-2 
 resulted in formation of nh2y in only 14  
 1% conversion , compared with 39% in the single mutant . furthermore , 
 the rate constant for its formation was decreased 10
fold 
 for the double mutant : from 12  1 to 0.13  0.01 s and only a single kinetic phase was measured .
both double mutants were also characterized 
 by 9 ghz epr spectroscopy in samples frozen at 30 s ( figure s4 ) . in the case of both double mutants ,
the altered 
 kinetics of pathway radical formation and its altered amplitude in 
 the case of nh2y730/c439a-2 
 are likely reflective of the importance of these h bonding interactions 
 in tuning of pcet within 2 . to test for possible conformational 
 differences between the nh2y intermediates in the 
 double vs the single mutants
, we measured their inter spin distances 
 to the diagonalized y122 by peldor spectroscopy 
 ( figure s5 and s6 ) .
the experiments revealed 
 distances of 3.8 and 3.9 nm , respectively , consistent within error 
 ( 1  ) with the distances observed in the single mutants 
 and from the 22 docking model in wt ; however , the assignment 
 of the 3.9 nm distance in the mutant nh2y730/c439a is more uncertain due to the low radical yield 
 and s / n ratio of the peldor traces hf 
 epr spectra ( 263 ghz ) of nh2y trapped in the double 
 mutants also revealed gx values ( figure s7 ) different from those 
 in the single mutants , i.e. , shifted by + 0.30.4 ppt in the 
 direction of a free nh2y as expected after removal 
 of a h bond ( table 1 ) .
h - endor 
 spectra of both double mutants ( figure 3b ) 
 display that the sharp peaks at 0.60.7 mhz have dramatically 
 decreased . in the spectrum of nh2y731/y730f the peaks
are now entirely absent , whereas residual peaks 
 ( 30% of initial intensity ) are observed in nh2y730/c439a mutant .
these results are consistent 
 with the assignment of one h bond to nh2y731 associated with y730 and one hydrogen bond to 
 nh2y730 associated with c439 .
importantly , the spectrum of nh2y731/y730f gives no evidence for any additional 
 strong h bonds .
the nh2y730/c439a mutant , on the other hand , still reveals a contribution of a strong 
 hydrogen bond presumably from y731 as previously proposed 
 by dft calculations .
we note also that 
 the removal of the hydrogen bond is manifested in the hf coupling 
 of the amino deuterons , which slightly decreases as compared to the 
 single mutants due to a change in the spin density distribution on 
 nd2y. to obtain a model structure 
 of the radical localized 
 at nh2y731 , dft calculations on representative 
 model systems for nh2y731 were performed 
 and the obtained magnetic parameters for gx and for h bonds were compared with the experimental 
 values . as a starting point
, we used the dft energy optimized structure 
 of y731 that was previously reported ( methods ) .
three large 
 models ( up to 216 atoms , figure s8 ) were 
 considered , which differed by the inclusion of zero , one or two water 
 molecules .
the models have taken into account all residues in an interaction 
 sphere of about 5  around the oxygen of y731 .
dft optimized structures of nh2y731. 
 left : model 1 includes the water molecule wat1 .
model 3 contains a second water molecule wat2 , 
 which is observed in some x - ray structures ( see text ) .
we found that the presence or 
 absence of the water molecules has 
 an impact on some residues arrangement around nh2y731 but not on the formation of a strong h bond with 
 y730 .
in optimized models 1 and 2 , the distance from the 
 oxygen of nh2y731 to the hydrogen of 
 the phenol group of y730 is 1.7  . in absence of wat1 
 ( model 2 ) ,
r411 approaches nh2y731 with hydrogens from its guanidinium group at distances of 
 2.1 and 2.6  from the oxygen of nh2y731. the calculated gx values from the two models differ by about 0.5 ppt , with the gx values of model 2 being closer to the 
 experimental 
 ones ( table 1 ) . in model 3 a second water molecule 
 ( wat2 )
was included as observed in some x - ray structures of wt- 
 and nh2y731/730- ( figure s9 ) in the vicinity ( ro  o  2.63.6  ) of residue 731 .
the optimized structure 
 of model 3 finds wat2 within hydrogen bond distance ( ro  h 1.9  ) to nh2y731 ; however , a stronger h bond to the phenoxyl hydrogen 
 of y730 remains ( ro  h 1.6  ) .
the gx value for the model 3 structure is consistent with the experimental 
 value ( table 1 ) .
the computed epr parameters 
 for the h bond distances and orientations relative to y730 are listed in table 2 .
particularly , all 
 models display an orientation of the strong h bond to nh2y731 provided by y730 , which is compatible 
 with the endor data within an uncertainty of about 20% ( table 2 , figure s10 ) .
parameters 
 were obtained from simulations 
 of the orientation selective 94-ghz endor spectra and comparison with 
 the dft models .
the signs of the couplings from the simulation are 
 only relative to each other within one tensor .
the euler angles (  , 
  ,  ) are defined from the a or q to the g tensor based on the y convention ( positive sign for a rotation is counterclockwise , 
 second rotation is around the y axis ) .
the a and q ( quadrupole ) tensor are chosen 
 such that |ax| < |ay| < |az| .
within this definition , for both 
 the amino deuterons and the h bond deuteron the ax direction results along the bond direction . 

euler angles from dft ( in the orca output positive rotations are defined 
 clockwise ) were transformed into the magnetic resonance convention , 
 for comparison .
uncertainty in the parameters from the dft and endor 
 simulations is estimated up to about 20% .
considering the accuracy of the calculations , on the 
 order of 0.5 
 ppt , all three models give gx values in principle compatible with the experiment .
however , 
 considering that the error in the trend of the calculation is smaller 
 than 0.5 ppt , model 2 and 3 better reproduce the experimental g values .
the results indicate that the strong gx shift at nh2y731 can be contributed by the combined effect of a strong h 
 bond to y730 and the interaction either to a water molecule 
 ( model 3 ) or alternatively to the positively charged arginine r411 ( model 2 ) .
the findings are further supported by calculations 
 on small model systems ( figure s11 ) , in 
 which the effect of the individual residues r411 or y730 was systematically tested .
both scenarios represented in 
 models 2 and 3 find some precedents in the literature .
studies on 
 cation interactions revealed that these are common 
 between amino acids like arginine and aromatic amino acids like tyrosine .
other studies on small peptides showed that tyrosine / arginine interactions 
 can alter the reduction potential of tyrosines . on the other hand , in ps ii
two water molecules have been 
 proposed to interact with the redox active yz during pcet 
 and affect their g values .
we note that 
 protons of either wat2 or r411 possibly located at distances 
 1.9  from the phenoxy oxygen might not be resolved in 
 an endor spectrum .
finally we note that the g values 
 for these nh2y can also be affected by the  
 subunit , which can not be modeled at present .
the electronic and structural features of the nh2ys 
 trapped in the rt pathway of e. coli rnr report on 
 how the local protein environment has reorganized after a pcet step 
 to stabilize these intermediates . at each radical state , the protein 
 has rearranged to accommodate a released electron and a proton , a 
 scenario that can be probed by two epr parameters : the shift in the gx value caused by positive 
 charges and the hyperfine interaction of protons forming hydrogen 
 bonds of varying strengths .
the first parameter is directly accessible 
 from high - frequency epr spectra but contains 
 only indirect information on hydrogen bond interactions .
the second 
 parameter , i.e. , the hyperfine coupling to protons exchanged by deuterons , 
 requires endor spectroscopy and much more extensive analysis , which 
 in turn , when attainable , uniquely delivers a high resolution structure 
 of the h bond partners . in this study
we report both parameters for 
 the nh2ys trapped within the rt pathway of rnr 
  subunit .
all gx values of the nh2ys in the three pathway positions 
 are similar and show a considerable shift from the calculated value 
 for a free nh2y ( table 1 ) , consistent with a strongly perturbed electrostatic environment . 
 however , our data reveal that the number , orientation and strength 
 of exchangeable hydrogen bonds at these residues are intrinsically 
 different , reflecting the subtle difference in protein architecture 
 and giving direct insight into the hydrogen bond network involved 
 in pcet . the endor data display
one strong hydrogen bond and 
 one moderate 
 h bond with nh2y730 and one strong hydrogen 
 bond with nh2y731. in both cases , the 
 hyperfine couplings and tensor orientations of the two strong bonds are very similar ( tables 2 and s2 ) and indicate that the hydrogen must reside 
 between the two tyrosyl rings ( figures 3 and 4 ) .
the direction of the hydrogen bond to nh2y731 extracted from the present hyperfine 
 and quadrupole tensors in the endor data ( figure 4 ) are consistent with the directions predicted by the dft 
 calculations for a bond with y730 ( figure 5 ) no matter the number of water molecules considered in the 
 calculation .
moreover , the endor peaks ( figure 3b ) of this strong bond disappear when y730 is mutated 
 to f730 . these results taken together provide strong evidence 
 for a direct hydrogen bond from y730 to nh2y731 and strongly suggest that the proton is directly 
 transferred via colinear pcet , as was proposed by previous quantum 
 chemical calculations .
thus , the results support the 
 current model for pcet ( figure 1 ) , that is , 
 that y731 functions as a proton acceptor for the 
 subsequent rt step to y730 .
they reveal that in each 
 case , the h bond must be directed almost perpendicular to the tyrosyl 
  system to reproduce the size of the hyperfine tensors and 
 the gx shift of each 
 nh2y. the results together provide strong support 
 for the  stacking of y731/y730 in the 
  active 
it should be pointed 
 out that this stacking is observed in some , but not all of the  resting2 
 structures ( yeast and nh2y 
 structures show residue 731 in multiple 
 conformations ) and that currently no atomic resolution structural 
 data are available for the complex . the unusual y / y  stacking 
 and the implications of this design relative to other configurations 
 for optimum efficiency of transfer has been studied by theorists .
the work of kaila and hummer indicates 
 that this configuration results in strong electronic coupling and 
 adiabatic , colinear pcet , consistent with our data .
finally , 
 the x - ray structure of the resting states of the wt , nh2y730- and nh2y731-s and our previous dft optimized structure of nh2y730 and y730-2 , show that residue c439 is in hydrogen 
 bond distance to y730 ( figure 1c ) .
the present experiments with the double mutant nh2y730/c439a corroborate 
 the assignment of c439 as a second moderate hydrogen bond 
 partner of y730. thus , the most direct interpretation 
 of our data combined with the calculated reduction potentials in ref ( 25 ) .
is that residue y730 acts as the direct hydrogen bond acceptor for c439 . in principle
it still remains possible that water does provide 
 the h - bond , and that the c439a mutant disrupts the h - bonding 
 pattern and eliminates it ; however , our experimental data provide 
 no evidence for the participation of a water in this proton transfer 
 step , in contrast to a recent proposal of bu et al . this group using dft and qm / mm calculations on several 
 model systems , in which cys could transfer a proton to y including 
 one with the  real protein  environment for c439 , y730 , suggested that the conserved water molecule 
 ( h2o138 in their notation is wat1 here ) moves 
 and inserts itself between these residues promoting a double proton - coupled electron transfer step .
however , their calculated 
 barrier for direct pcet between c439 and y730 of 60 kcal / mol is inconsistent with ours and siegbahn and co - workers calculations , which delivered a barrier of 89 kcal / mol . 

intriguingly , in one of their model systems with a cys and tyr located 
 on different peptide chains mimicking 
 the rnr configuration , the barrier for pcet is almost identical ( 9 
 kcal / mol ) to our data in the real system .
the studies with nh2y731/y730f ( figure 3b ) failed to reveal a h bonding 
 interaction with a second proton as observed with nh2y730-hsc439 .
however , in the center of the h - endor spectrum , the broad matrix line could be indeed contributed 
 by one or several weak h bonds , which are not resolved .
nevertheless , 
 the dft calculation revealed that r411 is capable of rearranging 
 to form weak hydrogen bonds ( 2.1  ) if it is not sterically 
 hindered by water molecules , and that this configuration , along with 
 the strong h bond to y730 , can reproduce the observed gx value .
however , a model with 
 two waters ( figure 5 ) , also is capable of recreating 
 the experimentally measured gx value .
the absence of structural insight about the / 
 subunit interface and the ability to use the protein environment ( r411 or two waters ) to subtly alter the electrostatic environment , 
 suggests multiple factors contribute to gx in addition to the strong h bond and our methods 
 can not currently distinguish between them .
perhaps the most 
 unexpected observation from the current studies 
 are the results with nh2y356-2 . 

in contrast with the pathway residues in 2 , no exchangeable 
 moderate h bonds to nh2y356 are observed 
 outside the matrix line , yet the gx value is perturbed to a similar extent as those for the nh2ys in 2 .
our inability to observe crystallographically 
 y356 within 2 alone or in 22 prevents 
 any specific conclusions about the origin of the gx shift .
however , the data still contribute 
 to our understanding of pcet at this position .
for example , one possible 
 mode of communication between y356 and y731 could 
 involve  stacking similar to that observed between y731 and y730 .
this type of interaction is unlikely , however , 
 given the absence of any moderate h bond .
a second 
 possible model of communication for pcet between y356 and 
 y731 , could involve a water molecule or network of water 
 molecules at the subunit interface .
the amino protons of nh2y356 would likely obscure these weak h bonds as 
 they are adjacent to the matrix line , precluding their detection . 

the greatly perturbed gx value of nh2y356 in the absence of 
 strong or moderate h bonds requires an altered electrostatic environment 
 provided by the protein . whether this environment could be provided 
 by water clusters at the interface or perhaps by binding 
 of mg ,
long been known to 
 play an important , but still poorly defined role in / , 
 / and  structure / chemistry , potentially in the interface region of the active complex , 
 requires further analysis .
in conclusion , the present 
 data establish a hydrogen bond network 
 between residues 731730439 in the 2 subunit . 

the observed hydrogen bonds and directions provide very strong support 
 for a colinear pcet mechanism , consistent with the recent finding 
 that the turnover rate constants within  are very fast , > 14000 
 s , when conformational gating from  is 
 removed by photoinitation .
these results 
 differ dramatically from the pcet process at the / interface 
 and within . our data indicate that colinear pcet and  
 stacking between y356 and y731 are unlikely , 
 and also reveal the importance of the electrostatic protein environment . 

additional spectroscopic experiments could be informative , but structural 
 insight is also essential . within 2
the combination of protein 
 engineering , spectroscopic data , and quantum chemical calculations 
 has provided much insight into the pcet process within this subunit . 

nature appears to have utilized multiple pcet strategies to achieve 
 this long - range oxidation over 35  .
adenosine-5-triphosphate 
 ( atp ) , cytidine-5-diphosphate ( cdp ) , reduced -nicotinamide 
 adenine dinucleotide phosphate ( nadph ) , hydroxyurea ( hu ) , kanamycin 
 ( km ) , chloramphenicol ( cm ) , 2xyt media , m9 minimal 
 salts , l - arabinose ( ara ) , -mercaptoethanol ( -me ) , 
 streptomycin sulfate and nh2y were purchased from sigma - aldrich . 
 isopropyl--d - thiogalactopyranoside ( iptg ) and 1,4-dithiothreotol 
 ( dtt ) were purchased from promega .
nucleotide primers 
 were purchased from invitrogen and pfu ultra ii polymerase 
 was purchased from stratagene .
( his)6-wt-2 
 ( 2200 nmol / min / mg ) and wt-2 ( 7000 nmol / min / mg and 1.2 y 
 /2 were expressed and purified by standard protocols .
all 2 mutants were prereduced 
 with 30 mm dtt and 15 mm hu before use.e .
coli thioredoxin ( tr , 40 u / mg ) and thioredoxin 
 reductase ( trr , 1800 u / mg ) used in assays were isolated as previously 
 described .
( his)6-nh2y730-2 and ( his)6-nh2y731-2 were purified as previously described . the quikchange kit ( stratagene ) 

the template pet - nrda with the appropriate stop codon 
 was amplified with primers 1 , 2 , and 3 and their reverse complements 
 were used to insert a ttt ( phe ) at position 730 ; gcc ( ala ) at position 
 439 and agc ( ser ) at position 439 ( table 3 ) .
all constructs 
 contain an n - terminal ( his)6-tag with a 10 amino acid linker , 
 as described previously .
expression and purification 
 of nh2y730-2 followed previous protocols except that the purification buffer ( 50 mm tris , 
 5% glycerol , 1 mm pmsf , ph 7.6 ) contained 1 mm tcep .
-nh2y730 , -nh2y731 , -nh2y356 and their double mutants were combined with 
 the corresponding wt( / ) 1:1 at final complex concentrations 
 of 100200 m in d2o and h2o assay 
 buffer as previously described .
the reaction was initiated 
 at 25 c by adding cdp and atp with final concentrations of 2 
 and 6 mm , respectively , and manually freeze quenched after 1020 
 s inside the epr tube with liquid n2 .
echo - detected epr 
 spectra at 263 ghz were recorded on a bruker elexsys e780 quasi optical 
 spectrometer using a single mode ( te011 ) cylindrical resonator 
 ( bruker biospin ) with a typical quality factor of 5001000 . 

maximum microwave power coupled to the resonator was about 15 mw . 
 the electron spin echo ( /2echo ) 

the 
 epr spectrum of y122 in wt- was used as 
 a reference ( figure s1 ) at 250 m 
 concentration in assay buffer to calibrate magnetic field .
samples 
 for 263 ghz epr were inserted in capillaries ( 0.33 od , vitrocom cv2033s / q ) 
 in typical volumes of 50 nl . samples for 94 ghz spectroscopy 
 contained typical volumes of 2 l in 0.9 mm od capillaries . 

samples frozen in liquid nitrogen were loaded into the resonator immersed 
 in liquid nitrogen and then transferred into the precooled epr cryostat .
94 ghz pulse epr and endor spectra were recorded on a bruker e680 
 w - band spectrometer with 400 mw microwave output power ( bruker power 
 upgrade 2 ) .
94 ghz h mims endor ( /2/2rf/2 
 echo ) was carried out using random radio frequency 
 irradiation and 40 s rf pulses produced with a 250 w rf - amplifier 
 ( 250a250a , amplifier research ) . all
derivatives were obtained 
 by fitting every four points with a second order polynomial and differentiating 
 the function in matlab ( version 7.10 ) .
the line width was set to 3 g with a line 
 broadening contribution of a 1:1 gaussian to lorenzian .
94-ghz h endor 
 spectra were simulated by using a matlab routine developed in house 
 that is based on a first order hamiltonian ( high field condition ) 
 for the hyperfine and quadrupolar interaction .
the blind spots produced 
 by the mims endor sequence were included by multiplying the calculated 
 endor powder pattern with an envelope function given by iendor = 1  cos(2a ) .
this is valid for the i = 1 
 nuclei considered here , as all quadrupole couplings are much smaller 
 than the hyperfine values .
the 
 initial model structures were based on the large models 7 and 8 used in ref ( 25 ) augmented by the amino group at y731 .
geometry optimizations have been performed using the bp86 gradient 
 corrected density functional in combination with 
 ahlrichs tzvp basis set of triple- quality .
the resolution of the identity approximation 
 with the corresponding auxiliary basis sets has been employed throughout . 

cartesian constraints were imposed on the position c of y730 , y731 and c439 as well 
 as c and c of all surrounding 
 residues .
additionally the cartesian coordinates of the hydrogen atoms 
 in the truncated gpd model replacing the bonds between c4 and c5 of 
 the ribose as well as the bond between c1 of the ribose and the base 
 were kept fixed .
the epr calculations and geometry 
 optimization of the small models were carried out with the b3lyp hybrid density functional in 
 combination with the rijcosx approximation . 
 here only the dihedral angle of the peptide bond of y730 and y731 was fixed and the cartesian restrains for all 
 surrounding c s were kept . in the small models 
 a solvation model ( cosmo ) with polarity 
  = 24 was used to compensate protein influences . for all dft / epr 
 calculations ,
barone s epr - ii ( iglo - ii for sulfur ) basis set 
 of double- quality has been used in combination with the def2-tzvpp / jk 
 auxiliary basis set for all atoms .
 ribonucleotide 
 reductases ( rnrs ) catalyze the conversion of ribonucleotides 
 to deoxyribonucleotides in all organisms . in all class 
 ia rnrs , initiation 
 of nucleotide diphosphate ( ndp ) reduction requires a reversible oxidation 
 over 35  by a tyrosyl radical ( y122 , escherichia coli ) in subunit 
  of a cysteine ( c439 ) in the active site of subunit 
 . this radical transfer ( rt ) occurs by a specific pathway involving 
 redox active tyrosines ( y122  y356 in 
  to y731  y730  c439 in  ) ; each oxidation necessitates loss of a proton 
 coupled to loss of an electron ( pcet ) . to study these steps , 
 3-aminotyrosine 
 was site - specifically incorporated in place of y356- , 
 y731- and y730- , and each protein was 
 incubated with the appropriate second subunit ( ) , cdp 
 and effector atp to trap an amino tyrosyl radical ( nh2y ) 
 in the active 22 complex . high - frequency ( 263 ghz ) pulse 
 electron paramagnetic resonance ( epr ) of the nh2ys 
 reported the gx values 
 with unprecedented resolution and revealed strong electrostatic effects 
 caused by the protein environment . 
 2h electron  nuclear 
 double resonance ( endor ) spectroscopy accompanied by quantum chemical 
 calculations provided spectroscopic evidence for hydrogen bond interactions 
 at the radical sites , i.e. , two exchangeable h bonds to nh2y730 , one to nh2y731 
 and none to nh2y356. similar experiments 
 with double mutants -nh2y730/c439a and -nh2y731/y730f allowed 
 assignment of the h bonding partner(s ) to a pathway residue(s ) providing 
 direct evidence for colinear pcet within . the implications 
 of these observations for the pcet process within  and at the 
 interface are discussed . 
Introduction
Results
Discussion
Conclusion
Methods