electrostatic 
 interactions have been shown to play a vital role 
 in enzyme catalysis . these interactions assist in the binding of ligands 
 and facilitate the enzyme - catalyzed chemical reactions by keeping 
 the reacting species positioned properly as well as stabilizing the 
 transition state in some cases . moreover , these electrostatic interactions are expected to be significantly 
 modulated by the conformational changes that occur over the catalytic 
 cycle . in particular , the electrostatic environment in the 
 active site is likely to change as the ligands bind , the chemical 
 reaction occurs , and the ligands are released . quantifying the electrostatic 
 contributions to the individual catalytic steps , as well as the impact 
 of conformational changes on the electrostatic interactions , is a 
 challenging yet important goal in the field of enzyme catalysis . to gain a better understanding of the catalytic role of electrostatics 
 and conformational motions , we probed the changes in the active site 
 electrostatic environment resulting from structural rearrangements 
 that occur along the catalytic cycle of escherichia 
 coli dihydrofolate reductase ( ecdhfr ) . this enzyme catalyzes the nadph - dependent conversion of 7,8-dihydrofolate 
 ( dhf ) to 5,6,7,8-tetrahydrofolate ( thf ) . x - ray crystallographic , nuclear 
 magnetic resonance ( nmr ) , theoretical , and photophysical studies have 
 shown that ecdhfr exhibits significant structural 
 rearrangements along the catalytic cycle . thus , ecdhfr is a highly suitable 
 system for studying how conformational changes can affect the active 
 site electrostatic environment , which can then modulate enzyme catalysis . recently , thiocyanate and other vibrational probes have been incorporated 
 into a number of biological systems to investigate the local electrostatic 
 environments . boxer and co - workers have explored the use of nitrile ( cn ) probes 
 to detect differences in the electric field projected along the cn 
 axis in proteins upon ligand binding , protein folding , and protein  protein 
 association . using this method , they showed that the electrostatic 
 environment in the active site of -3-ketosteroid 
 isomerase ( ksi ) exhibits limited rearrangement upon photoexcitation 
 of a ligand , where this excitation was proposed to mimic the movement 
 of charge during the catalyzed chemical transformation . the authors concluded that the ksi active site 
 is electrostatically preorganized for catalysis in its tertiary protein 
 structure and that major rearrangement is not necessary for catalysis . this observation is not unexpected due to the relatively rigid structure 
 of ksi that limits substantial conformational mobility , 
 thereby limiting electrostatic rearrangement . however , calculations 
 have shown that further active site reorganization , manifesting as 
 small conformational changes that position the reacting species for 
 chemistry , is still required in ksi to enable the catalyzed proton 
 transfer reactions . in contrast to ksi , 
 which exhibits limited protein motion along the catalytic cycle , ecdhfr is known to undergo significant conformational changes 
 along the catalytic cycle , suggesting that larger changes in electrostatics 
 could play a more significant role in all steps of the catalytic cycle . to examine the electrostatic interactions within the active site 
 of ecdhfr , we incorporated thiocyanate probes at 
 site - specific locations . using vibrational and c nmr 
 spectroscopic techniques , as well as computer simulations , we analyzed 
 the electrostatics and degree of hydration of the microenvironments 
 surrounding the probes . the electrostatic interactions arising from 
 the active site microenvironments surrounding the small , catalytically 
 nonintrusive probes led to different observed cn vibrational stretching 
 frequencies and c nmr chemical shifts along the catalytic 
 cycle . experimental data clearly show changes in the active site electrostatic 
 environment due to structural rearrangements . further insights were 
 obtained through mixed quantum mechanical and molecular mechanical 
 ( qm / mm ) analyses of classical molecular dynamics ( md ) simulations . these calculations were able to reproduce the experimentally measured 
 vibrational frequency shifts reported by the probes across the enzyme - catalyzed 
 hydride transfer step of the catalytic cycle . moreover , 
 the electrostatic contributions from the active site microenvironments 
 surrounding the probe were decomposed into the major components ( individual 
 residues and ligands ) . in addition , the experimentally validated theoretical 
 model allowed us to examine the electric field along the hydride transfer 
 pathway between the substrate and cofactor , a property that is catalytically 
 important yet unattainable experimentally . this integrated approach 
 provides valuable insights into the interplay between electrostatic 
 and conformational changes along the catalytic cycle of ecdhfr . 7,8-dihydrofolate 
 ( dhf ) and [ ( 4r)-h]nadph 
 ( nadpd ) were prepared 
 according to previously described procedures . -nicotinamide 
 adenine dinucleotide phosphate reduced tetra(cyclohexylammonium ) salt 
 ( nadph ) , -nicotinamide adenine dinucleotide phosphate hydrate 
 ( nadp ) , potassium cyanide - c ( 99 atom % c ) , methotrexate , methotrexate - agarose , dithiothreitol ( dtt ) , 
 folic acid ( 97% ; fol ) , sodium phosphate monobasic monohydrate 
 ( 98102% ) , and 5,5-dithiobis(2-nitrobenzoic acid ) ( ellman s 
 reagent ) were purchased from sigma - aldrich and used without further 
 purification . sodium phosphate dibasic 
 heptahydrate and 2-nitro-5-thiocyanatobenzoic acid ( 97% ; ntcb ) were 
 purchased from emd and tci america , respectively . ph values were measured 
 using an accumet model 13 - 620 - 300 standard combination electrode calibrated 
 with vwr certified standard aqueous buffers ( ph = 4 , 7 , and 10 ) . all 
 of the experiments , including kinetics and spectroscopic measurements , 
 were done in ph 7.0 50 mm sodium phosphate buffer unless otherwise 
 specified . first , the two native cysteines ( c85 and 
 c152 ) in wild - type ( wt ) ecdhfr were mutated to ala 
 and ser , respectively , to generate 
 cys ecdhfr using the stratagene quikchange 
 site - directed mutagenesis kit and the wt ecdhfr template 
 as described elsewhere . primer sequences 
 were 5-c gaa gcc atc gcg gcg gct ggt 
 gac gta cca g-3 ( c85a ) and 5-c tcg cat agc tat tca ttc gaa atc ctc g-3 ( c152s ) . the choice of 
 amino acid substitution was based on a previous report showing that 
 the c85a / c152s substitution on ecdhfr induced minimal 
 perturbation to the enzymatic activity . selective incorporation of cysteine was achieved through subsequent 
 mutations using the following primers : 5-ca atc ggt agg cct tgc ccc ggc cgc aaa aat att atc-3 ( l54c ) and 
 5-g att atg ggg cgc cat tgc tgg gaa 
 tca atc g-3 ( t46c ) . plasmid construction , protein expression , 
 and purification of mutant dhfrs were performed according to published 
 protocols . labeling of the single - cysteine ecdhfr constructs 
 ( cys l54c and cys t46c , denoted as l54c - cn and t46c - cn , 
 respectively ) was achieved using a modified version of a published 
 procedure . inside an amicon ultra-4 10k 
 nominal molecular weight limit ( nwml ) centrifugal filter tube , approximately 
 400 m of each ecdhfr construct was mixed with 
 four times excess of ntcb in 4 ml of 0.1 m sodium phosphate buffer 
 at ph 7.4 . the mixture was allowed to react at room temperature for 
 10 min , and reaction progress was monitored by the appearance of the 
 phenolic product using a uv  vis spectrophotometer . the protein 
 mixture was then concentrated by spinning at 5k rpm in a centrifuge 
 at 4 c . the protein sample was then washed twice with 12 mm 
 kcn in ph 7.0 50 mm sodium phosphate buffer , with a concentration 
 step after each wash . this was followed by washing the protein sample 
 with 50 mm ph 7.0 sodium phosphate buffer two more times before concentrating 
 the protein solution down to 0.8 ml , which was then applied 
 onto a size exclusion column ( econo - pac 10dg ; bio - rad ) that was pre - equilibrated 
 with ph 7.0 50 mm sodium phosphate buffer . the isolated labeled proteins 
 were concentrated in an amicon ultra-4 10k nmwl centrifugal filter 
 tube . the intact masses of scn labeled and unmodified samples were 
 confirmed by esi - ms , which yield deconvoluted m / z values of 17965 ( l54c)/17978 ( t46c ) and 17940 ( l54c)/17953 
 ( t46c ) da , respectively . the theoretical molecular weights for the 
 unmodified samples are 17941 ( l54c)/17953 ( t46c ) da . similar 
 preparation procedures were used for the scn labeled 
 samples . a mixture containing 10 mm ellman s reagent and 0.5 
 m kcn in ph 7.0 0.1 m sodium phosphate buffer was prepared 
 at room temperature for 15 min . this mixture was then allowed to react 
 with a sample of the unlabeled protein in an amicon ultra-4 10k nmwl 
 for 10 min at room temperature . the final concentrations of a typical 
 reaction mixture were 500 m protein , 5 mm ellman s reagent , 
 and 0.25 m kcn . the subsequent purification and isolation 
 steps were similar to those for scn labeling , except that kcn was used for washes rather than kcn . successful labeling was confirmed 
 spectroscopically by the release of one equivalent of the phenolic 
 product as well as c nmr comparison with the unlabeled 
 proteins . the pre - steady - state 
 hydride transfer reaction was monitored on an applied photophysics 
 stopped - flow spectrophotometer thermostated at 25 c . all of 
 the enzymatic reactions were performed in 50 mm sodium phosphate buffer 
 ph 7.0 . one of the syringes in the stopped - flow analyzer was loaded 
 with 20 m enzyme , 250 m nadph , and 2 mm dtt . after combining dhfr 
 and nadph as described above , the mixtures were prepared on ice for 
 5 min prior to the onset of the chemical reaction . upon mixing , the 
 final concentrations of the individual species in the reaction chamber 
 were halved . the conversion of dhf to thf was followed by the loss 
 of fluorescence resonance energy transfer from the enzyme to nadph . the reaction mixture was excited at 290 nm , and the emission was measured 
 using a 400 nm cutoff output filter . the 
 measure of absorbance vs time trace ( of burst phase ) was fit to standard 
 single exponential decay to obtain the hydride transfer rate ( khyd ) . the kinetic isotope effect ( kie ) for the 
 hydride transfer rate was also determined under the same experimental 
 conditions , with the exception that nadpd was used instead of nadph 
 in comparative experiments . steady - state enzyme turnover kinetics 
 ( kcat ) were studied following similar 
 experimental conditions , with the exception that the reaction progress 
 was monitored at 340 nm . under each condition , at least 5 independent 
 kinetic runs were conducted , and the average rate constants were used 
 for analysis . ftir spectra were 
 obtained at room temperature using a mattson research series ftir 
 spectrometer ( madison instruments , inc . ) equipped with a liquid - nitrogen 
 cooled hgcdte ( mct ) detector ( infrared associates , inc . ) . cyanylated 
 dhfr samples ( [ protein ] = 1.72.5 mm ) were mixed with appropriate 
 ligands ( 1:10 mol equiv ) in aqueous buffer and placed in a liquid 
 cell with two caf2 windows separated by a 56 m teflon 
 spacer . all nitrile ( cn ) stretch absorbance spectra for cyanylated 
 dhfr samples consisted of 2000 scans and the data were collected with 
 1.0 cm resolution . the cn stretch absorbance spectra 
 for cyanylated dhfr samples were obtained by subtracting a spectrum 
 of the aqueous buffer solution from the protein spectrum . the baseline 
 for each spectrum was corrected by fitting a fifth - order polynomial 
 fit to the data , as done previously by fafarman et al . and mcmahon et al . the fifth - order polynomial local fit functions were obtained by 
 defining roots at least 15 cm from the peak maximum , 
 meaning the experimental data and fits were forced to be equal at 
 points more than 15 cm from the central frequency . all published spectra are the averages of two independently prepared 
 protein samples after the baseline has been corrected and the peak 
 height has been normalized to one . the peak positions obtained from 
 independently prepared protein samples typically differed by < 0.5 
 cm , which is substantially less than the variation 
 among peak positions due to the protein binding or releasing the ligands . 
 detailed experimental setup and data analysis are provided in the supporting information . c nmr spectra were 
 collected on a bruker av - iii-600 ( 150.9 
 mhz c frequency ) at 25 c . samples were prepared 
 with 1.6 mm of the scn labeled protein , 8 mm ligands , 
 and 50 mm potassium phosphate ( ph 7.0 ) in water . a sealed capillary 
 tube containing 1 m acetone in d2o was inserted into the 
 nmr tube that holds the protein sample . the 1d c 
 nmr spectral data obtained for each sample were compared with its 
 corresponding c dept 45 ( distortionless enhancement 
 by polarization transfer ) data to confirm the identity of the assigned 
 peaks . under the same experimental conditions , we were unable to detect 
 the c nmr signals corresponding to the scn probes for 
 samples containing either the nonisotopically enriched scn label or 
 the unlabeled proteins . crystals 
 of e. coli t46c - cn and l54c - cn dhfr 
 variants were obtained by the hanging - drop vapor diffusion method 
 using conditions similar to those previously described . detailed methods , data collection , and refinement 
 statistics are provided in the supporting information . we calculated 
 the ir spectra for the cn stretching mode of the incorporated thiocyanate 
 probes using a two - step method . first we propagated classical md trajectories 
 for the entire solvated enzyme with bound ligands . the configurations 
 sampled from the md trajectory were then analyzed using a series of 
 qm / mm single point energy calculations to determine the cn stretching 
 potential energy curve . the qm region was treated with a semiempirical 
 method that was reparameterized for the calculation of nitrile vibrational 
 frequencies , and the mm region was represented as a collection of 
 atomic point charges . the instantaneous vibrational wave functions 
 and associated frequencies from the energy level splittings were obtained 
 from the cn stretching potential energy curve using a grid - based numerical 
 approach . the simulated ir spectra were calculated using the fluctuating 
 frequency approximation , which includes the dynamical contributions 
 of the protein motion to the simulated lineshapes . a full description of the methodology can be found in our 
 previous application to ksi , with some 
 minor adjustments that will be discussed below . we propagated classical 
 md trajectories for the cysteine - free t46c - cn and l54c - cn ecdhfr mutants using the gromacs program . the first state has folate ( fol ) bound 
 in the substrate pocket and the nicotinamide adenine dinucleotide 
 phosphate cofactor bound in the oxidized form ( nadp ) . 
 in this state , the met20 loop is in the closed conformation , which 
 is thought to facilitate the hydride transfer reaction by positioning 
 the ligand and cofactor while excluding solvent molecules . this state has been shown to structurally mimic 
 the reactive michaelis complex ( e : dhf : nadph ) prior to hydride transfer . although we can simulate the reactive michaelis 
 complex without hydride transfer occurring , we simulated the e : fol : nadp ternary complex to compare directly with the experimentally 
 measured ir spectra . the second state that we simulated has the product 
 thf and nadp bound . in this state , the met20 loop is in 
 the occluded conformation , and the nicotinamide ring of nadp is excluded from the active site . these two states are used to model 
 the closed to occluded transition that occurs across the hydride transfer 
 step of the ecdhfr catalytic cycle . the initial 
 structures for the md simulations were obtained from crystal structures 
 solved with the nitrile probes included for both the l54c - cn and t46c - cn 
 variants ( pdb accession codes 4p68 and 4p66 , respectively ) . the crystal structures 
 have methotrexate bound in the substrate binding pocket and nadph 
 bound in the active site . in both of these structures , this state is thought to mimic the 
 transition state for the hydride transfer reaction . for the simulations in the closed conformation ( nadp / fol ) , the folate molecule was incorporated by aligning the 
 folate substrate with the corresponding heavy atoms of the methotrexate 
 ligand from the crystal structure . for the simulations in the occluded 
 conformation ( nadp / thf ) , we used the initial coordinates 
 of a wt crystal structure of ecdhfr complexed with 
 nadph and 5,10-dideazatetrahydrofolate ( ddthf ) ( pdb 1rx6 ) . the thiocyanate probes were incorporated into the occluded 
 structure by aligning the backbone atoms of the five previous and 
 five subsequent residues to the cyanylated cysteine residue in the 
 new crystal structure that contains the probe to the corresponding 
 backbone atoms of the 1rx6 structure . we then replaced the specific residue with 
 the aligned cyanalated cysteine and optimized the probe residue geometry 
 and position using a steepest - descent algorithm in gromacs , while 
 the ligands , the crystallographic water molecules , and the rest of 
 the enzyme were held fixed . the substrate ligand ( ddthf ) was replaced 
 by thf by aligning the corresponding heavy atoms in ddthf and thf , 
 and the oxidized cofactor was modeled by aligning the heavy atoms 
 of nadp with the nadph from the crystal structure . the 
 modeled ligands were also optimized using the steepest - descent algorithm 
 with the enzyme and the crystallographic water molecules held fixed . for all of the md simulations , we used the amber99sb force field 
 to compute the potential energy and forces for the protein . the bonded and nonbonded parameters for the nadp cofactor were consistent with those from our previous 
 work . the fol and thf charges were calculated 
 using the restrained electrostatic potential ( resp ) method , and the bonded and van der waals parameters 
 were obtained from the generalized amber force field ( gaff ) . for methyl thiocyanate and the cyanylated cysteine 
 residue , the bonded parameters were obtained from cho et al . , and the nonbonded parameters were obtained from 
 lindquist and corcelli , as used in our 
 previously published work on ksi . each of the enzymatic systems was solvated with tip3p water in a truncated octahedral box , where the sides 
 of the box were at least 10.0  from every atom in the enzymatic 
 complex , including the crystallographic waters . after solvation , we 
 added 12 sodium atoms using the gromacs genion utility 
 to neutralize the system before simulation . all bonds involving a 
 hydrogen atom were constrained to their equilibrium bond lengths using 
 the lincs algorithm as implemented in gromacs . nonbonded van der waals interactions were calculated with 
 a cutoff of 1.0 nm , and electrostatic interactions were calculated 
 using the particle - mesh ewald method . after the initial preparation of each system , we followed 
 a well - defined 
 equilibration procedure . the solvent configurations were minimized 
 using the steepest - descent algorithm in gromacs with the enzyme and 
 ligands held fixed to mitigate any possible bad contacts between the 
 solvent and solute molecules . the solvent molecules and counterions 
 were then equilibrated for 50 ps at 300 k in the canonical ( constant 
 nvt ) ensemble with the protein and ligands still fixed . after solvent 
 equilibration , the entire system was then minimized using the steepest - descent 
 algorithm . subsequently , the system was equilibrated using a simulated 
 annealing procedure in which the initial velocities were generated 
 according to a boltzmann distribution at 50 k. the system was equilibrated 
 in the isothermal  isobaric ensemble ( constant npt ) at 50 k 
 for 50 ps , and then the temperature was increased up to 300 k in increments 
 of 50 k with 50 ps of equilibration at each temperature . after this 
 simulated annealing procedure , an additional 1 ns of equilibration 
 in the constant npt ensemble was performed , and finally 1 ns of equilibration 
 in the constant nvt ensemble was performed . during the simulations , the nos  hoover thermostat was used to maintain the temperature , and the parrinello the production trajectories were propagated in 
 the canonical ensemble 
 at 300 k with a time step of 1 fs and a total simulation time of 1 
 ns for each independent trajectory . configurations were saved at every 
 time step and were analyzed using the qm / mm methodology described 
 below . for each mutant and catalytic state , we propagated at least 
 two independent trajectories with different initial configurations . the data for a single trajectory are presented in the main paper , 
 while data from additional trajectories are provided in the supporting information . the results for independent 
 trajectories associated with the same system were found to be consistent 
 within the numerical errors of the methodology . the methodology 
 used to calculate the vibrational frequency of the 
 nitrile group is similar to that used in our previous work . here we highlight the differences between the 
 methods used in our previous work and the methods employed in the 
 present work . as in our previous work , we calculated the vibrational 
 frequency by generating the one - dimensional potential energy curve 
 along the cn stretching mode using a qm / mm method . the potential energy is calculated along the cn vibrational coordinate by freezing all 
 of the atomic centers in the system and moving only the nitrogen atom 
 to stretch the cn distance from 1.0 to1.4  with a step size 
 of 0.05  . the qm calculations were performed with the mm region 
 treated as atomic point charges that polarize the qm region . these 
 atomic point charges were the same as those used in the md simulations 
 and were obtained from the amber99sb force field for the standard 
 amino acid residues , the tip3p force field for the water molecules , 
 and the resp calculations for the ligands . the choice of the qm region 
 for each probe studied in this work will be discussed below . in our 
 previous work , the one - dimensional potential 
 energy curve for the cn stretching mode was fit to a morse function , 
 and the energy levels were calculated analytically from the morse 
 parameters . in the present work , the potential energy curve was fit 
 to a third - order b - spline potential , and 
 the vibrational wave functions and energy levels were calculated numerically 
 using the fourier grid hamiltonian ( fgh ) method . an advantage of the fgh method is that it does not assume any functional 
 form of the potential energy curve and therefore avoids the bias associated 
 with fitting to the morse function , which depends on only three parameters . in principle , any electronic structure method can be used to calculate 
 the vibrational stretching potential , but the level of theory must 
 be computationally tractable for the millions of configurations that 
 are required to obtain statistically converged ir spectra . as in our 
 previous work , we used a reparameterized version of the semiempirical 
 pm3 method . in this semiempirical method , 
 some of the computationally expensive integrals in hartree these empirical parameters can be varied to reproduce 
 properties from either electronic structure theory calculations or 
 experimental measurements . because we altered our approach to use 
 the fgh numerical method to calculate the vibrational frequencies , 
 we reoptimized the pm3 parameters . the reparameterization procedure 
 is similar to that from our previous studies , and a full description 
 is provided in the supporting information . we also confirmed that the new set of parameters reproduces the 
 results for ksi from our previous work when the fgh numerical method 
 is used for calculating the vibrational frequencies . table s6 provides the calculated frequencies for the two different 
 vibrational probes in ksi that were studied previously . to assist 
 in the analysis of the vibrational frequency calculations , we calculated 
 the average electric field at the midpoint of the cn bond in the thiocyanate 
 group from our md simulations . the vibrational stark effect between 
 two states is described by1here h is 
 planck s 
 constant , cn is the shift in vibrational 
 frequency between the two states , eenv is the difference in the electric field vector at 
 the nitrile bond as a result of the change in the environment , cn is the difference dipole moment 
 for the vibrational mode that lies along the cn bond axis , and  
 is the angle between these two vectors . the magnitude of cn is the stark tuning rate , which 
 is a measure of the susceptibility of the probe to changes in the 
 local electric field . boxer and co - workers have measured the stark 
 tuning rate for thiocyanate probes in various environments , including 
 in proteins , to be 0.7 cm/(mv / cm ) . we emphasize that this equation accounts only 
 for the classical electrostatic effects of the environment and does 
 not account for the quantum mechanical aspects of effects such as 
 specific hydrogen - bonding interactions and polarization . while these 
 phenomena have significant classical components , they also have nonclassical 
 aspects that can be incorporated into the calculations by treating 
 interacting residues quantum mechanically , as will be discussed below . recent work explores the role of a local field correction factor that scales the externally applied electric 
 field to obtain the local field experienced by the probe , which is 
 typically larger because of the polarization of the local environment 
 induced by the applied electric field . this local field correction 
 factor has been estimated to be between 1.1 and 1.3 for protein environments , but recent investigations have suggested that it may be as large 
 as 1.8 . we have not considered the role 
 of the local field correction factor in our analysis of the electric 
 fields in dhfr due to the lack of consensus about its value for a 
 protein environment . moreover , applying the local field corrections 
 would result in changes that are within the error bars of our calculations . to calculate the electric field at the nitrile bond , a virtual 
 particle was positioned at the midpoint of the cn axis during the 
 md trajectory . this virtual particle had no electrostatic or van der 
 waals interactions with other particles , and the bond , angle , and 
 dihedral parameters were set to zero . the configurations from the 
 md trajectory were saved , and subsequently the forces acting on all 
 particles were recalculated for these configurations when the charge 
 on the virtual particle was set to + 1 . the electric field , evp , at the virtual particle location 
 was calculated as2where fvp is the force acting on the virtual particle and qvp is the charge of the virtual particle . the total electric 
 field vector , as well as the component along the cn axis , was calculated 
 for each configuration along the md trajectory . calculation of the 
 component of the electric field along the cn axis allows us to calculate 
 the change in the vibrational frequency due to classical electrostatics 
 using eq 1 . the remaining frequency change can 
 be attributed to the quantum mechanical aspects of specific hydrogen - bonding 
 interactions and polarization effects . these effects can be investigated 
 by using a classical polarizable force field or by treating the interacting residues 
 quantum mechanically . note that the experimentally measured and theoretically 
 calculated frequency shifts can be compared directly , but the breakdown 
 into the classical electrostatics and quantum mechanical aspects is 
 available only from the theoretical calculations . we also analyzed 
 the contributions of individual residues and ligands 
 to the electric field at the midpoint of the cn bond . because the 
 classical electric field is the sum of the individual contributions 
 from each atom , the electric field analysis of the contributions 
 from specific residues provides insight into the impact of conformational 
 changes occurring during the closed to occluded transition associated 
 with hydride transfer . we generated 
 the t46c - cn and l54c - cn mutants by site - specifically incorporating 
 a thiocyanate probe at each position ( figure 1 ) . the t46c - cn probe is situated in close proximity to the junction 
 between the two reacting ligands , dhf and nadph , allowing us to monitor 
 the microenvironment close to the reaction center . the l54c - cn probe is located 9  from the 
 center of the hydride donor  acceptor ( d  a ) axis . this 
 probe monitors the folate binding pocket , and its spectral character 
 is expected to respond to the formation of the ternary michaelis menten 
 complex upon folate binding , as well as the release of thf in the 
 enzyme turnover step . ( a ) superposition of the t46c - cn and l54c - cn ecdhfr mutants in the closed conformation with folate and nadp bound , where only the thiocyanate residue is shown for the 
 l54c - cn mutant . ( b ) ecdhfr in the closed ( red ) and 
 the occluded ( blue ) conformations exhibited by the ( c ) five major 
 complexes in its catalytic cycle . the enzyme activity of the two ecdhfr variants 
 was evaluated by their ability to catalyze the nadph - dependent reduction 
 of dhf and the enzyme turnover rates at ph 7.0 and 25 c . the 
 catalytic efficiencies of the t46c - cn ( khyd = 120  10 s ; kcat = 6.5  0.5 s ) and l54c - cn ( khyd = 250  20 s ; kcat = 28  3 s ) variants were 
 comparable to that of wt ecdhfr ( khyd = 220 s ; kcat = 12 s ) . we also found 
 the cys ecdhfr ( c85a / c152s ; khyd = 215  8 s ; kcat = 10.3  0.8 s ) variant to 
 exhibit activity similar to the wt enzyme . furthermore , figure 
 s1 shows that the crystal structures of the ternary complexes 
 of the t46c - cn and l54c - cn mutants in the closed conformation overlap 
 well with the wt crystal structure . these observations suggest that 
 the incorporation of the scn probes into the selected locations of 
 the active site does not significantly alter the structure or activity 
 of the enzyme . it should be noted that we also incorporated 
 the scn probe at the 
 i50 position ( cys - i50c - cn ) . however , such a modification decreased 
 the hydride transfer rate by 10 times at ph 7.0 and 25 c . we felt that the degree of enzyme activity reduction in cys - i50c - cn 
 might result in a less accurate representation of the electrostatic 
 environment in the active site of the wt enzyme . thus , further investigations 
 were not performed for the cys - i50c - cn construct . it is expected 
 that even small modifications to the crowded active site can have 
 large effects on enzyme activity . the ecdhfr 
 catalytic cycle contains the five major species ( figure 1c ) that reflect the significant conformational changes in 
 the active site met20 loop ( figure 1b ) . menten 
 complex ( e : nadph : dhf ) to an occluded conformation in the initial ternary 
 product complex ( e : nadp : thf ) . the enzyme remains in the 
 occluded conformation in the two subsequent product - containing species 
 ( e : thf and e : nadph : thf ) , until the met20 loop reverts back to the 
 closed conformation upon the rate - limiting release of thf to generate e : nadph ( figure 1c ) . nmr relaxation dispersion experiments have found that the rates 
 of the met20 loop conformational change are temporally similar to 
 their corresponding enzymatic processes . in other words , the hydride transfer and enzyme turnover rates are 
 similar to the values determined from the nmr experiments for the 
 conformational transitions that connect the relevant enzyme complexes . 
 while the major change in the met20 loop conformation appears to be 
 induced by the formation of the product thf , theoretical simulations have shown that minor structural rearrangements 
 of the ligands and protein , not restricted to the met20 loop , are 
 necessary to progressively optimize the substrate orientations and 
 the active site environment along the reaction coordinate of the hydride 
 transfer reaction . herein , we utilized the scn probes 
 to experimentally monitor the changes in the local microenvironments 
 in two locations in the active site over the five major catalytic 
 species . the five complexes that constitute the catalytic cycle 
 of ecdhfr ( figure 2 ) were 
 generated using 1.52.5 mm of the enzyme and at least five 
 times excess of the ligands in ph 7.0 phosphate buffer . these concentrations 
 should be more than sufficient to push the equilibria toward full 
 complexation , thus allowing us to obtain vibrational ftir spectra of the five 
 major complexes that constitute the ecdhfr catalytic 
 cycle , as well as for the apoenzyme and the e : nadph : fol complex . note 
 that the e : nadph : dhf state is modeled by e : nadp : fol , as 
 discussed in the literature . for each 
 enzyme complex , the ir spectrum was obtained in duplicate / triplicate 
 using separately prepared samples , and these spectra were found to 
 be very reproducible ( data in supporting information ) . after correcting the ir data for baseline and subsequently normalizing 
 the amplitude of the peak to 1.0 , we determined the maximum frequency 
 ( max ) from the first derivative of the corrected 
 ir peak . the peak position is a measure of the transition energy between 
 the vibrational ground state and first excited state of the thiocyanate 
 probe . the transition energy is sensitive to the electric field along 
 the cn bond because the vibrational potential energy function is perturbed 
 by the local electrostatic environment ( surrounding residues , ligands , 
 and solvent molecules ) which modulates the energy difference between 
 the vibrational quantum states . color coding the frequency of maximum 
 absorption detected in each complex illustrates the changes in the 
 local electrostatic interactions experienced by the cn probe over 
 the catalytic cycle ( figure 2 ) . ( b ) 
 nmr ( scn ) measurements along 
 the catalytic cycle of ecdhfr . the color scales for 
 the ftir and nmr data have units of cm and ppm , 
 respectively . the cofactor is orange , and the substrate / product is 
 purple in all complexes ( the coloring of the cofactor and substrate / product 
 does not correspond to the color scales that represent the nmr shift 
 or ir frequency ) . enzyme complex labels in blue are in the occluded 
 conformation , and those in red are in the closed conformation . the 
 measured values for ir frequency and nmr chemical shift are provided 
 in table s4 . figure 2 indicates that the local 
 environment 
 of each cn probe changes significantly over several key steps in the 
 catalytic cycle : formation of the ternary michaelis menten 
 model ground state complex , the hydride transfer step , and the rate - limiting 
 release of thf . the largest frequency shifts observed for the t46c - cn 
 probe occurred across the two most important steps in the catalytic 
 cycle : ( 1 ) upon the conversion between the model michaelis menten 
 e : nadp : fol complex and the initial e : nadp : thf 
 product complex ( 4.1 cm ) , which is analogous 
 to the hydride transfer step ; ( 2 ) across the enzyme turnover step , 
 which is the release of thf ( 5.5 cm ) . for the 
 l54c - cn probe , the most significant change occurred upon the formation 
 of the ternary e : nadp : fol complex ( 2.4 cm ) . crystal structures of the wt enzyme have shown that the t46 and 
 l54 residues retain their positions and orientations in the five catalytic 
 complexes , suggesting that the inserted cn probes likely experience 
 limited freedom of movement in the active site . md simulations confirm that there are no major conformational 
 changes in the probe orientation during 5 ns trajectories . therefore , 
 the observed changes in the microenvironments as the system moves 
 through the various complexes in the catalytic cycle are likely due 
 to protein conformational rearrangements that induce changes in the 
 electrostatic interactions between the probes and their surroundings . note also that the redox state of the cofactor ( nadph / nadp ) has very little effect on both the ir and c nmr data 
 ( table s4 ) . in many cases , we also 
 observed changes in the peak width [ full - width 
 at half - maximum ( fwhm ) ; table s4 ] , which 
 usually reflects the variation of chemical environments sampled by 
 the probe as well as the strength of intermolecular interactions such 
 as hydrogen bonding . for example , the l54c - cn probe , which is located 
 in the folate binding site , reported peak widths that are approximately 
 two times smaller in complexes with bound fol or thf . this observation 
 is consistent with the probe sampling a less fluctuating and more 
 well - defined electrostatic environment resulting from the various 
 interactions that organize the fol / thf to bind in the active site . there is also some evidence indicating that the e : nadph complex and 
 the apoenzyme exhibit a greater degree of hydrogen bonding between 
 a solvent water molecule and the l54c - cn probe . one indication of 
 this phenomenon is obtained from inspection of the c 
 nmr - ir relationship , which shows that the data for the above two complexes 
 deviate from the rest of the data in a manner that implicates greater 
 specific solvent hydrogen - bonding interactions ( figure s4 ) . md simulations support the presence of additional 
 solvent hydrogen - bonding interactions in these two complexes for the 
 l54c - cn probe , with a water molecule hydrogen bonded to the nitrile 
 probe in nearly half of the configurations . we also observed a slightly 
 broader ir peak in the e : nadph : fol complex , which can be attributed 
 to the slow reduction of fol over the course of the ir measurement . 
 this is not unexpected since the l54c - cn probe is located in the folate 
 binding pocket . these trends are less obvious for the t46c - cn probe , 
 which seems to be located in a more fluctuating environment , as indicated 
 by the relative ir peak widths . we also prepared the isotopically 
 enriched cys - cn 
 variants ( t46c - cn and l54c - cn ) to obtain 
 the complementary c nmr data for all of the studied complexes . 
 while the magnitude of the observed changes are different because 
 the c nmr chemical shift is less sensitive to specific 
 solvent hydrogen - bonding interactions than is the cn vibrational stretching 
 frequency , the c nmr data illustrate 
 similar trends to those shown by the corresponding ir data . in particular , 
 we observed a changing microenvironment around the scn 
 probes as the enzyme evolves through the catalytic cycle ( figure 2b ) . similar to the ir data , the microenvironment 
 detected at the t46c - cn probe exhibits larger changes 
 along the catalytic cycle than that at the l54c - cn probe . this trend is expected due to the proximity of the t46c - cn site to the regions of the substrates that undergo the hydride 
 transfer reaction . overall , the experimental data summarized as figure 2 clearly demonstrate a dynamic electrostatic environment 
 in the active site of ecdhfr across its catalytic 
 cycle . the differences in the microenvironments of the two probes 
 and the way they change among the different complexes , especially 
 between the closed and occluded conformations , in the ecdhfr catalytic cycle also provide an indication of the highly heterogeneous 
 nature of the enzyme active site . to identify 
 the various environmental contributions that give rise to the observed 
 cn stretching vibrational frequency shifts along the catalytic cycle , 
 we conducted qm / mm simulations to monitor the electrostatic interactions 
 experienced by the probes for the complexes along the catalytic cycle . these data provide information about how the microenvironments of 
 the probes reorganize across individual steps of the catalytic cycle . we simulated the ir spectra of the t46c - cn and l54c - cn mutants of ecdhfr for two different complexes along the catalytic cycle : 
 ( 1 ) the ternary complex with the reactant analog fol and nadp bound , which mimics the e : nadph : dhf state prior to hydride 
 transfer ; ( 2 ) the ternary complex with the product thf and nadp bound , which is the e : nadp : thf state following 
 hydride transfer . first we examined the local environment around the 
 t46c - cn probe , which is located near the midpoint between the two 
 bound ligands ( figure 1a ) . the qm region includes 
 the t46c - cn residue , the substrate ( either fol or thf ) , and the nearest 
 water molecule to the nitrile nitrogen , as depicted in figure 3a . the qm region was determined by calculating the 
 cn frequency including the entire probe microenvironment in the qm 
 region for a subset of the configurations sampled and comparing these 
 results to those obtained with smaller qm regions . the details of 
 the procedure used to determine the qm region are provided in the supporting information . configurations from md 
 simulations for the ( a ) t46c - cn and ( b ) 
 l54c - cn ecdhfr mutants . the residues with thicker 
 lines are included in the qm region for the qm / mm calculations of 
 vibrational frequencies . figure 4a presents the calculated 
 and experimentally 
 measured ir spectra for the t46c - cn mutant for both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . the calculated 
 ir spectrum for the closed conformation ( solid black curve ) has a 
 maximum at 2165.8 cm , which is within 1.8 cm of the measured peak in the experiments . the agreement 
 between the calculated and experimental absolute frequencies is potentially 
 fortuitous , but the level of agreement is similar to that calculated 
 in our previous studies of ksi . the fwhm 
 of the calculated ir spectrum for the closed conformation is 11 cm , which is in good agreement with the experimentally 
 measured line width of 14 cm . the calculated ir 
 spectrum for the occluded product state ( solid red curve ) has a maximum 
 at 2162.1 cm . the calculated shift between these 
 two states is 3.7 cm , which is in excellent 
 agreement with the experimentally measured shift of 4.1 cm . note that our md simulations and cn frequency calculations 
 are performed on the nanosecond time scale . the probe could experience 
 multiple additional conformations on the longer time scale associated 
 with the ir measurements . during our md simulations of 5 ns , no major 
 structural rearrangements of the probe orientation occurred , indicating 
 that we are only sampling a single conformational state . since the 
 starting configurations for our simulations were based on crystal 
 structures that include the probe , we expect to be sampling the most 
 stable and therefore the dominant configuration of the probe . moreover , 
 the agreement between the calculated and experimental cn frequency 
 shifts provides further validation of the methodology . calculated and experimentally 
 measured ir spectra of the cn vibrational 
 stretching frequency for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound ( closed conformation ; 
 black line ) and with nadp / thf bound ( occluded conformation ; 
 red line ) . the solid lines are the simulated spectra from the qm / mm 
 calculations , and the dotted lines are the measured spectra from the 
 ftir experiments . in addition to the ir 
 spectra , we calculated the electric field 
 at the midpoint of the cn bond for configurations sampled by the md 
 trajectory using the methodology described above . from these calculations , 
 we can correlate the measured vibrational frequency shifts to changes 
 in electrostatics from the protein environment . figure 5a depicts the calculated electric field along the cn axis 
 for the two states . the average projected electric field for the closed 
 ( nadp / fol ) and occluded ( nadp / thf ) states 
 are 15.9 and 19.0 mv / cm , respectively . using the experimental 
 stark tuning rate of 0.7 cm/(mv / cm ) in conjunction 
 with eq 1 , the difference of 3.1 mv / cm 
 corresponds to a shift in the vibrational frequency of 2.2 
 cm . the difference between this frequency shift 
 arising from only classical electrostatics and the actual shift of 
 ca . 4.0 cm obtained from both calculations 
 and experiments is attributed to the quantum mechanical aspects of 
 hydrogen - bonding interactions and polarization effects that are not 
 included in the linear relation given by eq 1 . calculated electric field along the cn bond at the midpoint of 
 this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr 
 systems with nadp / fol bound ( closed conformation ; black 
 line ) and with nadp / thf bound ( occluded conformation ; 
 red line ) . previously it has been shown experimentally and computationally that significant deviations of 
 10 cm from the vibrational frequency shifts 
 predicted by eq 1 can occur when the nitrile 
 probe participates in specific hydrogen - bonding interactions . according 
 to our md simulations , the t46c - cn thiocyanate probe is solvent accessible 
 in both the closed and occluded conformations and directly hydrogen 
 bonds to a water molecule , as depicted in figure 3a . in both protein conformations , the nitrile probe is also 
 able to act as a hydrogen - bond acceptor from the amine group in the 
 substrate ( fol / thf ) backbone . note that the observation of hydrogen - bonding 
 interactions with the probe for these two states is consistent with 
 the experimental ir - nmr correlation data in figure 
 s4 . thus , the difference between the frequency shift 
 of 2.2 
 cm predicted from eq 1 , 
 which includes only classical electrostatic effects , and the shift 
 of 3.7 cm obtained from the qm / mm calculations 
 of the full system can be attributed to hydrogen - bonding interactions 
 with the water molecule and polarization effects from the water molecule 
 and the substrate ( fol / thf ) . to confirm this hypothesis , we performed 
 the qm / mm frequency analysis with only the probe residue ( t46c - cn ) 
 in the qm region ( i.e. , switching the water molecule and the substrate 
 from the qm region to the mm region ) . in this case , the calculated 
 frequency shift between the two states was 2.6 cm , which agrees well with the 2.2 cm shift 
 obtained from eq 1 . beyond the calculation 
 of the total electric field at the probes , 
 we can decompose the field along the cn axis into contributions from 
 individual residues in the enzymatic complexes . in essence , this methodology 
 allows us to decompose the microenvironment surrounding the cn probe 
 into the constituent parts , which then allows us to identify the major 
 contributing components . for the t46c - cn probe ( figure 3a ) , the sum of the contributions from the main contributors 
 given in table 1 corresponds to a change in 
 electric field along the cn axis of 2.9 mv / cm , which is consistent 
 with the change of 3.1 mv / cm calculated with the entire enzyme . the contributions from the residues in the closed conformation 
 are shown pictorially in figure 6a , where the 
 residues that are colored blue contribute positively and those that 
 are colored red contribute negatively to the total field along the 
 cn bond . for the t46c - cn probe , the nadp cofactor and 
 the nearest water molecule are the dominant contributors to the change 
 in the electric field along the cn axis , although asn18 and ser49 
 also make significant contributions ( table 1 ) . the presence of a positive charge on the nicotinamide ring and 
 the short 4  distance between the nitrile nitrogen and 
 the aromatic nitrogen in the nicotinamide ring lead to a strong contribution 
 to the electric field in the closed conformation . after the transition 
 to the occluded conformation , the cofactor is excluded from the active 
 site , and the magnitude of the contribution to the field from the 
 nicotinamide ring is much smaller due to the larger distance of 14 
  between the two nitrogen atoms . this effect of the movement 
 of nadp on the electric field at the probe demonstrates 
 how atomic motions can significantly alter the local electrostatics 
 in the active site . moreover , the 3.2 mv / cm change in the 
 electric field contributed by asn18 and ser49 is consistent with the 
 structural rearrangement from the closed to the occluded conformation . specifically , the amide nitrogen of asn18 is 1.5  closer 
 to the nitrile probe in the closed conformation than in the occluded 
 conformation , and the hydroxyl group of ser49 is 1.0  
 closer to the nitrile probe in the closed conformation . contributions 
 to the calculated electric field along the cn bond 
 at the midpoint of this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound and the met20 
 loop in the closed conformation . the color for each residue corresponds 
 to the calculated values in tables 1 and 2 , respectively , using the color scale provided , 
 although residues contributing a magnitude > 4.0 mv / cm are depicted 
 in the darkest color . the residues that contribute negatively to the 
 field are colored red , the residues that contribute positively to 
 the field are colored blue , and the residues that have no net contribution 
 to the field along the cn bond are colored white . changes 
 in the electric field along 
 the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . additionally , following the closed 
 to occluded transition , the 
 cn probe becomes more accessible to the solvent . the contribution 
 to the electric field along the cn axis from the closest water molecule 
 changes by 7.46 mv / cm during this transition . moreover , the 
 sign of the average electric field along the cn axis due to this water 
 molecule is different for the closed and occluded conformations , with 
 a value of 2.14 mv / cm for the closed conformation and 5.32 
 mv / cm for the occluded conformation . analysis of the md trajectories 
 indicates that a water molecule is hydrogen bonded to the nitrile 
 probe 26% of the time in the closed ( nadp / fol ) 
 conformation and 52% of the time in the occluded ( nadp / thf ) conformation . the positive contribution 
 to the electric field along the cn axis from the nearest water molecule 
 in the closed conformation is due to configurations in which the nearest 
 water molecule has its negatively charged oxygen atom pointing toward 
 the nitrile . these configurations are stabilized by the hydrogen - bonding 
 interaction of the water molecule with the hydroxyl group of ser49 , 
 as shown in figure s5 . the negative contribution 
 to the field in the occluded conformation arises from configurations 
 in which a hydrogen atom of the nearest water molecule is oriented 
 toward the probe , forming a hydrogen bond between the water molecule 
 and the cn group . thus , this computational approach is also able to 
 probe the detailed active site hydration status , which is not easily 
 accessible with experimental means . we performed the same analysis for the l54c - cn mutant 
 to examine the microenvironment near the folate binding pocket . figure 4b depicts the calculated and experimentally measured 
 vibrational spectra for the l54c - cn mutant in both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . the l54c - cn 
 probe is distal from the active site , and it is located in a relatively 
 hydrophobic pocket near the carboxylate tail of the substrate , as 
 shown in figure 1 . the qm region used for this 
 mutant includes the l54c - cn residue , phe31 , and arg57 , as depicted 
 in figure 3b . the qm region was determined 
 by calculating the cn frequency including the entire probe microenvironment 
 in the qm region for a subset of the configurations sampled and comparing 
 these results to those obtained with smaller qm regions . the details 
 of the procedure used to determine the qm region are provided in the supporting information . the calculated frequency 
 shift between the two states for the l54c - cn probe is 2.5 
 cm , which is in reasonable agreement with the 
 shift of 1.0 cm measured experimentally . figure 5b depicts the calculated electric 
 fields along the cn bond for the l54c - cn mutant in the two states . the calculated average electric fields along the cn probe in the closed 
 and occluded conformations are 8.4 and 13.8 mv / cm , 
 respectively . using the experimental stark tuning rate of 0.7 cm/(mv / cm ) in conjunction with eq 1 , this shift of 5.4 mv / cm corresponds to a shift in vibrational 
 frequency of 3.8 cm due to only classical 
 electrostatic interactions . to model the situation with only classical 
 electrostatic interactions , we calculated the shift in the vibrational 
 frequency when only the residue containing the thiocyanate group ( l54c - cn ) 
 is treated quantum mechanically and the remainder of the system , including 
 phe31 and arg57 , the calculated 
 frequency shift when only the probe residue is treated quantum mechanically 
 is 3.5 cm , which is consistent with the 
 value of 3.8 cm obtained from eq 1 . however , this frequency shift is greater than the 
 shift of 2.5 cm calculated with the larger 
 qm region . the greater shift predicted by the purely classical electrostatic 
 model indicates that the polarization effects due to the phe31 and 
 arg57 residues decrease the vibrational frequency shift relative to 
 that predicted by classical electrostatics . table 2 provides the contribution to the 
 electric field along the cn axis for several key residues near the 
 l54c - cn probe . the sum of these contributions is 7.3 mv / cm , 
 which is slightly higher than the change of 5.4 mv / cm calculated 
 with the entire enzyme , indicating that other residues are also contributing 
 to this field . in a similar manner to the analysis for the t46c - cn 
 mutant , the arg57 residue exhibits the largest change 
 between the two states with a net shift of 6.58 mv / cm . in 
 the closed conformation , the line connecting the midpoint of the cn 
 bond to the protonation site of the positively charged arg57 is nearly 
 perpendicular to the cn axis , resulting in almost zero contribution 
 to the electric field along the cn axis . the change in the electric 
 field contribution by arg57 is likely due to a geometrical shift that 
 moved the positive charge on arg57 away from this orthogonal orientation 
 with respect to the probe in the occluded conformation . interestingly , 
 while the substrate exerts a large contribution to the electric field 
 along the cn axis , this contribution remains nearly constant between 
 the two states . the stable orientation of the cn axis relative to 
 the negatively charged carboxylate moiety in the bound substrate results 
 in negligible change in the contribution of the substrate to the electric 
 field at the cn probe . changes 
 in the electric field along 
 the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . while the calculated electric 
 fields along the cn axes of the probes provide a measure of the electrostatic 
 microenvironments within the active site , the electric field along 
 the hydride transfer d encouraged by the good agreement between the experimental 
 and the simulated data described above , we applied the same methodology 
 to evaluate the total electric field at the midpoint of the d  a 
 axis and the field projected along the d  a axis in the closed 
 ( e : nadp : fol ) state . the negative value of the field indicates 
 that it points from the substrate to the cofactor and that this field 
 will facilitate the transfer of a negatively charged hydride from 
 the cofactor to the substrate . in other words , the projection and 
 the magnitude of the field along the d  a axis in the ternary 
 michaelis complex is highly favorable for the hydride transfer reaction 
 to occur . the substrate and cofactor contribute a total of 32.4 
 mv / cm to the field along the d  a axis . the rest of the protein 
 contributes approximately an additional 31% ( 15.4 
 mv / cm ) of the total field . also , the solvent molecules and ions contribute 
 an additional 1.1 mv / cm to the field along the d  a 
 axis . this analysis suggests that a major function of the enzyme 
 is to bring the substrate and cofactor into a configuration that realizes 
 the highly favorable field for facilitating the hydride transfer reaction 
 ( figure 7 ) . in the absence of the enzyme , it 
 would be less probable for the reacting species , nadph and dhf , to 
 achieve the same relative orientation in solution . moreover , this 
 analysis provides a new perspective on the role of electrostatic catalytic 
 contributions in dhfr . it is currently impossible to experimentally 
 measure the electric field along the d  a axis , but the calculations 
 provide reasonable estimates . a favorable electric field of 15.4 
 mv / cm from the protein and 32.4 mv / cm from the ligands along 
 the d  a axis could potentially reduce the free energy barrier 
 by several kcal / mol . while these values 
 suggest a substantial catalytic contribution generated from the active 
 site electrostatic environment , it is important to acknowledge that 
 the full mechanism of catalysis is far more complex than a simplistic 
 evaluation based solely on the electric field along the d  a 
 axis . depiction of the component of the electric field along the hydride 
 transfer d  a axis calculated from an md simulation of wt ecdhfr with nadp / fol bound and the met20 loop 
 in the closed conformation . the color for each residue corresponds 
 to the calculated electric field values using the color scale provided , 
 although residues contributing a magnitude > 4.0 mv / cm are depicted 
 in the darkest color . the three arrows in the red oval represent the 
 total electric field of 48.9 mv / cm ( purple ) , the field of 
 32.4 mv / cm resulting from the ligands ( yellow ) , and the field 
 of 16.5 mv / cm resulting from the rest of the system ( green ) 
 projected along the d  a axis . we also decomposed this field into the contributions from 
 all of 
 the protein residues , and our analysis identified many residues that 
 contribute to the electric field along the d  a axis . figure 7 depicts the contributions from each of the residues 
 to the field along the d  a axis . the residues that are colored 
 blue contribute positively and those that are colored red contribute 
 negatively to the field pointing from the substrate to the cofactor 
 along the d  a axis . the residues that are shown with side chains 
 contribute to the field with a magnitude > 1 mv / cm . interestingly , there is a strong degree of overlap between the 
 residues that strongly contribute to the field along the d  a 
 axis and the network of coupled motions that has been identified for 
 dhfr catalysis . the residues ile14 , tyr100 , 
 and asp122 contribute 1.2 , 4.2 , and 2.4 mv / cm , 
 respectively , and were all implicated in the network of coupled motions . additional residues that strongly contribute to the electric field 
 along the d  a axis are lys32 ( 3.2 mv / cm ) , arg52 ( 3.9 
 mv / cm ) , and arg57 ( 4.2 mv / cm ) . the residues that contribute 
 strongly against the field along the d  a axis include asp27 
 ( 5.4 mv / cm ) , arg98 ( 3.4 mv / cm ) , and his124 ( 3.0 mv / cm ) . all of these 
 residues represent potential targets to alter the electrostatic contribution 
 to catalysis . we investigated 
 the changing electrostatic environment at two different 
 thiocyanate probes inserted into the active site of ecdhfr for five complexes along the catalytic cycle . our results illustrate 
 a changing electrostatic environment near the hydride transfer site 
 ( t46c - cn ) as well as a smaller change in key regions of the folate 
 binding pocket ( l54c - cn ) . the changes in electrostatic environment 
 are associated with conformational changes occurring along the catalytic 
 cycle of ecdhfr . experimental evidence for the changing 
 electrostatic environment includes both ir frequency and c nmr measurements , which interrogate the electric field along the 
 cn axis and the total electric field at the probe . these experimental 
 data demonstrate fundamental aspects of the electrostatic landscape 
 for catalysis in ecdhfr . molecular dynamics 
 simulations provided an atomic - level picture 
 of the local environment experienced by the thiocyanate probes in 
 complexes that model the states prior to and subsequent to the hydride 
 transfer reaction . the qm / mm vibrational frequency calculations reproduced 
 the experimentally measured frequency shifts for these two states , 
 which span the closed to occluded transition associated with hydride 
 transfer . analysis of the md trajectories provided insight into the 
 conformational changes occurring between these two states . the factors 
 leading to the vibrational frequency shifts were broken down into 
 classical electrostatic effects , specific hydrogen - bonding interactions , 
 and polarization effects . thus , the vibrational frequency shifts are 
 not simply a manifestation of the classical electric fields but rather 
 include additional contributions from the environment . moreover , the 
 electric fields along the cn axis of the probes were decomposed into 
 contributions from specific residues , ligands , and solvent molecules . the electric field along the hydride d  a axis was also calculated 
 with the experimentally validated methodology . the calculations illustrate 
 that the cofactor and substrate , as well as the enzyme , impose a substantial 
 electric field along the d  a axis that facilitates hydride 
 transfer . overall , experimental and theoretical data provide 
 evidence for 
 significant electrostatic changes in the microenvironments that constitute 
 the heterogeneous ecdhfr active site as the enzyme 
 progresses along the catalytic cycle . the electrostatic interactions 
 between the protein and the ligands assist in orienting the reacting 
 species in a geometry that maintains a large electric field favoring 
 hydride transfer from the cofactor to the substrate . the enzyme active 
 site environment also provides a significant portion ( 1/3 ) 
 of the total electric field that facilitates the hydride transfer 
 reaction , suggesting the catalytic importance of enzymatic conformational 
 changes that alter the active site electrostatics . future work will 
 investigate the five trapped states along the catalytic cycle for 
 the two probes studied in this work as well as additional probe sites 
 in the ecdhfr system to provide a comprehensive picture 
 of the electrostatic landscape for the entire catalytic cycle .
electrostatic interactions play an 
 important role in enzyme catalysis 
 by guiding ligand binding and facilitating chemical reactions . 
 these 
 electrostatic interactions are modulated by conformational changes 
 occurring over the catalytic cycle . 
 herein , the changes in active 
 site electrostatic microenvironments are examined for all enzyme complexes 
 along the catalytic cycle of escherichia coli dihydrofolate reductase ( ecdhfr ) by incorporation 
 of thiocyanate probes at two site - specific locations in the active 
 site . the electrostatics and degree of hydration of the microenvironments 
 surrounding the probes 
 are investigated with spectroscopic techniques 
 and mixed quantum mechanical / molecular mechanical ( qm / mm ) calculations . 
 changes in the electrostatic microenvironments along the catalytic 
 environment lead to different nitrile ( cn ) vibrational stretching 
 frequencies and 13c nmr chemical shifts . 
 these environmental 
 changes arise from protein conformational rearrangements during catalysis . 
 the qm / mm calculations reproduce the experimentally measured vibrational 
 frequency shifts of the thiocyanate probes across the catalyzed hydride 
 transfer step , which spans the closed and occluded conformations of 
 the enzyme . 
 analysis of the molecular dynamics trajectories provides 
 insight into the conformational changes occurring between these two 
 states and the resulting changes in classical electrostatics and specific 
 hydrogen - bonding interactions . 
 the electric fields along the cn axes 
 of the probes are decomposed into contributions from specific residues , 
 ligands , and solvent molecules that make up the microenvironments 
 around the probes . 
 moreover , calculation of the electric field along 
 the hydride donor  acceptor axis , along with decomposition of 
 this field into specific contributions , indicates that the cofactor 
 and substrate , as well as the enzyme , impose a substantial electric 
 field that facilitates hydride transfer . 
 overall , experimental and 
 theoretical data provide evidence for significant electrostatic changes 
 in the active site microenvironments due to conformational motion 
 occurring over the catalytic cycle of ecdhfr .