oxalate decarboxylase 
 ( oxdc ) catalyzes the mn - dependent conversion 
 of oxalate monoanion into co2 and formate . although the enzyme appears to be found in many fungi and 
 some bacteria , its biological function 
 remains poorly understood .
it has been shown , however , that oxdc is 
 a constituent protein of the spore coat formed by bacillus 
 subtilis .
details of the mechanism by which oxdc mediates 
 cleavage of the chemically unreactive c  c bond in oxalate remain to be clearly delineated .
heavy - atom isotope 
 effect ( ie ) measurements and recent spin - trapping 
 experiments are consistent with the hypothesis 
 that reaction proceeds via heterolytic breakdown of a manganese - bound 
 oxalate radical anion . in principle
, the 
 presence of mn(ii ) in oxdc offers the opportunity to use epr methods 
 to explore the mechanistic role of the metal ion in catalysis .
the situation is complicated , however , by the 
 fact that the enzyme is composed of two cupin domains , each of which 
 contains a mn(ii ) ion in a similar coordination environment ( figure 1 ) . as a result , even 
 the high - field epr spectra of the wild - type enzyme in buffer are complicated , 
 and the effects of substrate binding and/or catalysis at a specific 
 metal center are difficult to observe .
it is therefore not surprising 
 that two prior experimental studies have yielded conflicting assignments 
 for the observed fine structure parameters of the two mn(ii ) centers .
representations of the mn(ii ) binding sites in the n - terminal 
 ( right ) 
 and c - terminal ( left ) domains of bacillus subtilis oxalate decarboxylase ( 1uw8 ) .
the location of the 
 two mn(ii ) centers in the monomer are indicated by the solid spheres 
 in the ribbon drawing ( top ) .
color scheme : c , cyan ; 
 n , blue ; o , red ; mn , purple .
the figure was rendered using vmd . in principle , the application 
 of modern theoretical methods to 
 obtain estimates of the fine structure parameters d and e associated with each of the two mn(ii ) centers 
 in oxdc represents a means of resolving the discrepancy in assignments 
 of the previous epr studies . in practice , however , any accurate computation 
 of fine structure parameters from first principles represents a significant 
 challenge for modern quantum mechanical methods .
this is especially 
 true for high - spin d systems where the balance between 
 the direct electron  electron magnetic dipole spin  spin 
 ( ss ) interaction involving unpaired electrons and a second order term , 
 arising from the spin  orbit coupling ( soc ) of electronically 
 excited states into the ground state , gives rise to very small d values ranging from nearly 0 to 1.5 cm ( corresponding to interaction energies on the order of 0.004 kcal 
 mol ) .
estimates of fine structure parameters obtained 
 using theoretical methods are therefore the difference between large 
 contributions that have the same order of magnitude and opposing signs . 

thus , even minor errors in calculating either of the ss or soc contributions 
 can significantly affect the accuracy of the final results .
efforts to employ density functional theory 
 ( dft ) for the accurate prediction of fine structure parameters are 
 also complicated by the delicate balance between exchange and correlation 
 contributions .
be that as it may , any demonstration that theoretical 
 calculations are capable of yielding even qualitative insights into 
 zero - field splittings would have wide application in the study of 
 mn - dependent enzymes and likely facilitate the interpretation of epr 
 spectra for multicenter systems , such as oxdc . to date , however , only one study has been published in which an 
 integrated experimental / computational strategy was used to assign 
 the fine structure parameters associated with mn(ii ) ions bound to 
 enolase .
we now report a mutational 
 strategy that has allowed us to establish 
 the assignment of epr fine structure parameters to each of the mn(ii ) 
 centers in wild - type oxdc , at least at high solution ph .
this work 
 has also provided an opportunity to assess the performance of a combined 
 dft and dft / mm computational strategy for calculating the zero - field 
 splitting ( zfs ) parameters of the protein - bound mn(ii ) ions in oxdc . 
 despite the known sensitivity of calculated d and e values to the computational approach ,
we demonstrate that good estimates of these parameters 
 can be obtained using cluster models taken from carefully optimized 
 dft / mm structures .
overall , our findings not only confirm the assignments 
 of tabares et al . but also provide new 
 insights into the strengths and limitations of theoretical methods 
 for understanding electronic properties of protein - bound mn(ii ) ions . 

this work also sets the stage for future epr studies on the electronic 
 properties of the mn(ii ) centers in oxdc and site - specific variants .
mn(ii ) is a d ion with a total 
 spin quantum number 
 of s = 5/2 .
the transition between the ms = 5/2 and ms = 3/2 
 electron spin manifolds is emphasized at temperatures below 4 k because 
 of a large population difference according to boltzmann statistics .
spectroscopically , this transition extends over 
 a field range of 6(d + e)/gb , whereas that of the next higher energy 
 transition between ms = 3/2 and ms = 1/2 , typically observed at temperatures 
 above 5 k , is only 3(d + e)/gb .
this 
 is also true for the central transition between ms = 1/2 and ms = + 1/2 , 
 although because these transitions are not directly affected by the 
 fine structure to first order they are typically very sharp and intense . 

importantly , frozen solution spectra observed at very low temperatures , 
 at or below 4 k , which feature the ms = 
 5/2 and ms = 3/2 transitions , 
 allow the direct determination of not only d and e values but also the sign of d. hence , the largest deviation of the powder 
 spectrum from the center happens at low field with the main turning 
 point at 4|d|/gb ( measured from the center of the spectrum ) for negative d values , whereas the highest field component appears at 
 ( 2|d| + 6|e|)/gb ( figure s1 in supporting 
 information ) .
the situation is reversed if d has a positive sign . in the event that the rhombic component , e , has an appreciable magnitude ,
a splitting is observed 
 on the side with the smaller deviation from the center , featuring 
 signals at ( 2|d| + 6|e|)/gb and ( 2|d|  
 6|e|)/gb .
the 
 sign of e depends on how one defines the x- and the y - axis of the fine structure 
 tensor , and it can only be determined using single - crystal epr spectroscopy . in studies of oxdc and its site - specific 
 variants ,
our strategy of resolving the fine structure relies on observing 
 the transitions between higher ms states 
 at the lowest possible temperature , which is between 2.5 and 3 k for 
 our helium flow cryostat .
these transitions are usually not very strong 
 and require enzyme concentrations on the order of 20 mg / ml or higher 
 to be reliably observed .
we also routinely take epr spectra at intermediate 
 temperature ( 40 k ) to distinguish between species exhibiting small 
 and relatively large magnitudes of the fine structure parameters d and e. the high - field epr ( 326.4 ghz ) 
 spectrum of wild - type ( wt ) oxdc at ph 8.5 in frozen solution ( 3 k ) 
 clearly showed transitions associated with higher ms states ( figure 2 ) , enabling us 
 to determine the fine structure values directly for at least one of 
 the two mn(ii ) species present in the enzyme .
this ph was chosen because 
 the x - ray crystal structure of wt oxdc , which was used in dft and 
 dft / mm calculations , was obtained by crystallization at ph 8.5 .
we 
 also note that observation of these weak transitions required the 
 use of high enzyme concentrations ( 27 mg / ml ) , which could be achieved 
 by modification of published purification protocols .
the intensity of the central |+1/2 
  |1/2 sextet centered at 11658 mt was greatly 
 reduced due to boltzmann population favoring the transition from the 
 lowest level within the electron spin manifolds , |5/2 
  |3/2. on its low - field side , a broad peak 
 was visible , centered at around 11450 mt , and on the high - field side , 
 there was a negative signal at 11760 mt .
a poorly resolved shoulder 
 was observed above 11800 mt instead of the expected rhombic splitting , 
 and because a fully axial fine structure tensor would yield a much 
 narrower peak at high fields , this implied that the fine structure 
 parameter , e , had a broad distribution spanning essentially 
 the whole range from zero to the maximum possible value of |d|/3 . by using a broad gaussian distribution of e centered around 200 mhz with a half - width of 800 mhz , 

however , we were able to obtain a reasonable fit of the experimental 
 spectrum ( figure 2 ) .
on the other hand , this 
 approach was unable to recover the high - field shoulder present in 
 the spectrum , implying that our experimental e / d ratios ( table 1 ) have large errors 
 associated with them , thereby making it difficult to draw hard conclusions 
 from these values .
high - field ( 326.4 ghz ) epr spectra of wt oxdc taken at 
 3 k ( black ) 
 and 40 k ( blue ) together with spectral simulations .
note that the 
 central part of the spectrum at 40 k together with the corresponding 
 spectral simulation of site i is removed for clarity . the enzyme ( 27 
 mg / ml ) was dissolved in 50 mm tris buffer containing 500 mm nacl , 
 ph 8.5 .
( site i ) with small zfs at 3 k ( red ) and 40 k ( green ) and 
 a single mn(ii ) species ( site ii ) with very large zfs at 40 k ( cyan ) . 

the sum of site i and site ii simulations at 40 k is also shown ( magenta ) . 

we note that site ii does not contribute significantly to the spectral 
 range visible . unedited spectra and full sets of simulation parameters 
 are provided elsewhere ( figures s2s4 in supporting information ) .
the error for e / d values obtained in the present study is very 
 large due to the broad distribution of e values that 
 had to be assumed in the simulations .
these values are obtained from 
 the center of a broad gaussian distribution of e ( see 
 text for more details ) .
cluster model containing hydroxide 
 as the fifth ligand ( figure 7c ) . cluster model containing hydroxide 
 as the fifth ligand with a hydrogen bond to a solvent water ( figure 7d ) .
initial 
 structure for qm / mm optimization 
 based on the x - ray crystal structure of co(ii)-containing w132f oxdc 
 variant .
initial structure 
 for qm / mm optimization 
 from in silico modification of the x - ray crystal structure of wt oxdc 
 ( 1uw8 ) . in the 
 text , we report either the 
 positive or the negative d value as obtained by calculation . 
 in this table , however , we give only absolute values for d in the case of the c - terminal site because the sign of this parameter 
 could not be experimentally determined .
the spectrum at 40 k primarily showed the |3/2 
  |1/2 transitions on the low- and high - field 
 side of a much stronger set of central |+1/2  |1/2 
 sextet lines relative to those seen at 3 k ( figure 2 ) .
these transitions were now located closer to the spectral 
 center , and the |5/2  |3/2 
 transitions were barely visible above the noise .
interestingly , there 
 were three relatively sharp transitions visible on the low - field side 
 of the spectrum together with a broader feature on the high - field 
 side as one would expect from a pentacoordinate species with 
 |d| of approximately 10 ghz .
the appearance of overlapping 
 spectra with differing zero - field splittings is consistent with the 
 fact that oxdc contains two mn binding sites .
simulations of the 40 k high - field epr spectrum using the site i 
 magnetic parameters extracted from the spectrum at 3 k together with 
 a second species ( site ii ) having very large fine structure ( 10 
 ghz ) gave a satisfactory global fit ( figure 2 ) and yielded similar values for the fine structure parameters d and e ( table 1 ) to those reported previously .
site 
 i has the smallest |d| value and carries about 68% 
 of the spectral weight . unfortunately , the transitions associated 
 with the second mn(ii ) ( site ii ) were not of sufficient intensity 
 to allow direct experimental observation .
instead , we had to rely 
 on the second - order effect of the fine structure on the central ms = 1/2 to ms = + 1/2 transitions , manifesting itself through broadenings and spectral 
 splittings , which turn the sextet lines into a group of sharp lines 
 at low field complemented by a broad pake pattern on the high - field 
 side ( figure s2 in supporting information ) . given that these are second - order 
 effects on the + 1/2  1/2 transitions ,
no information 
 concerning the sign of d could be obtained from our 
 simulation .
values for the g - factor and hyperfine 
 coupling constant a with the mn nucleus 
 were also obtained for the two mn(ii ) sites in the enzyme ( table s1 
 in supporting information ) .
both of these 
 sites are similar to ones reported by tabares and co - workers ( table 1 ) , with site ii most 
 probably corresponding to a pentacoordinate mn(ii ) ion observed 
 in the c - terminal domain of the enzyme by x - ray crystallography ( figure 1 ) .
hence , site i is 
 likely associated with a hexacoordinate mn(ii ) bound within 
 the n - terminal domain of the enzyme . in order to confirm 
 these assignments
, we decided to prepare a 
 site - specific oxdc variant in which only the zfs parameters of the 
 n - terminal mn(ii ) center would be perturbed , thereby leading to a 
 shift in the d and e values associated 
 with only one site .
an analysis of the second sphere interactions 
 revealed a hydrogen bonding interaction between the side chains of 
 a conserved tryptophan ( trp-132 ) and the glutamate ( glu-101 ) coordinating 
 mn(ii ) in the n - terminal domain ( figure 3 ) . 

we anticipated that removing this hydrogen bond would perturb the 
 mn(ii)/glu-101 interaction , thereby affecting the zfs parameters , 
 and so the w132f oxdc variant , in which trp-132 is replaced by phenylalanine , 
 was expressed and purified following our standard procedures .
steady - state kinetic analysis gave values 
 of 63 s and 50 mm for kcat and km , respectively ( figure s5 in supporting information ) , and so the w132f oxdc 
 variant exhibited a 10-fold lower catalytic efficiency ( kcat / km ) than wt enzyme after 
 normalizing for mn(ii ) incorporation .
however , the observations that 
 ( i ) kcat was essentially unaffected by 
 the site - specific mutation and ( ii ) mn(ii ) incorporation was very 
 similar in wt oxdc and the w132f oxdc variant supported the idea that 
 the tertiary structure was not significantly affected by removal of 
 the hydrogen bonding interaction .
schematic representation of hydrogen bonding 
 interactions ( dotted 
 lines ) involving residues in the bacillus subtilis oxdc n - terminal mn(ii ) binding site when the active site loop adopts 
 a  closed  conformation .
copyright 2004 american society for biochemistry and molecular 
 biology . in order to ensure 
 that there was indeed minimal impact on mn(ii ) 
 coordination and local protein structure as a result of replacing 
 trp-132 by phenylalanine , we sought to obtain an x - ray crystal 
 structure of this oxdc variant .
all efforts to grow diffraction quality 
 crystals of the mn(ii)-substituted form of the w132f oxdc variant 
 proved unsuccessful .
we were able , however , to express and purify 
 the co(ii)-containing form of the mutant enzyme using conditions that 
 had been developed in prior investigations into oxdc metal selectivity , and this material gave crystals that were suitable 
 for structure determination .
the x - ray crystal structure of co - substituted 
 w132f oxdc was solved using phases calculated by molecular replacement 
 and refined to a resolution of 2.1  .
the overall protein fold 
 of the co - containing w132f variant did not differ significantly from 
 wt enzyme , and the two structures superimposed with an rmsd of only 
 0.26  .
more importantly , perhaps , the phenyl ring in the oxdc 
 w132f variant was positioned in an identical orientation to the indole 
 side chain of trp-132 within the n - terminal mn(ii ) binding site ( figure 4 ) .
comparison of the metal  ligand bond distances 
 in the co - substituted form of the w132f oxdc variant and the mn - containing 
 wt enzyme also showed that they do not vary significantly in light 
 of a 0.23  coordinate error for the w132f structure ( figure 
 s6 in supporting information ) .
this similarity 
 of co(ii ) and mn(ii ) metal  ligand bond lengths in oxdc is consistent 
 with data in the scripps metalloprotein database .
for example , the average bond lengths for protein residues 
 to mn(ii ) ranges from 1.5 to 2.9  with an average value of 2.20 
  .
co(ii)-containing proteins have an average amino acid ligand 
 bond length of 2.16  and range from 1.55 to 2.69  .
similarly , 
 the bond distances for water ligands bound to mn(ii ) and co(ii ) range 
 from 1.05 to 3.09  ( 2.26  average ) and 1.362.99 
  ( 2.26  average ) , respectively .
hence , at least in terms 
 of metal  ligand bond distances and angles , co(ii ) is a good 
 mimic of mn(ii ) .
as importantly , and despite the fact that co - containing 
 wt oxdc is catalytically inactive , the 
 side chain carboxylate of the functionally important residue glu-162 , which is located on a mobile active site loop 
 segment ser - glu - asn - ser ( bacillus subtilis numbering ) , undergoes only a small shift in position as a result of the site - specific 
 mutation . on the other hand , we noted that the ring of his-97 had 
 become  flipped  relative to its orientation in wt oxdc 
 ( figure 5 ) .
efforts to refine the structure 
 of the variant enzyme with his-97 oriented in the same manner as observed 
 in wt enzyme , however , merely resulted in reorientation of the ring 
 to the original position in our initial structure for the w132f variant . 

the correct positioning of the his-97 side chain was also verified 
 by a composite omit map ( figure s7 in supporting 
 information ) .
importantly , the structure of the co - substituted 
 enzyme confirmed that replacement of trp-132 by phenylalanine 
 did not perturb the structure of the c - terminal mn(ii ) binding site 
 ( figure 4 ) .
schematic representation of the structural 
 changes in the n - terminal 
 ( top ) and c - terminal ( bottom ) metal centers resulting from replacement 
 of trp-132 by phenylalanine .
the figure was generated by superimposing 
 the x - ray crystal structures of wt oxdc ( 1uw8 ) 
 ( purple ) and the co - containing w132f oxdc variant ( green ) .
superimposition of the mn(ii ) n - terminal binding sites 
 observed 
 in the x - ray crystal structures of wt oxdc ( orange ) and the co - containing 
 w132f oxdc variant ( green ) . only the residues in the first coordination 
 sphere around the metal
figure rendered using 
 vmd . given the kinetic and structural evidence that removal of the hydrogen 

bonding interaction between glu-101 and trp-132 was likely only to 
 perturb the n - terminal mn(ii ) center , we measured the high - field epr 
 ( 326.4 ghz ) spectrum of the w132f oxdc variant at ph 8.5 ( figure 6 ) .
the w132f oxdc variant shows a broader spectrum 
 of its transitions between higher ms states when compared with the wt enzyme .
hence , the low - field 
 maximum now appeared at 11386 mt , and although the negative peak appeared 
 to be centered at 11780 mt ( little shifted compared to wt enzyme ) , 
 it was broader and reached out to higher fields ( figure 6 ) . as perhaps expected from the negligible alterations to 
 the structure of the c - terminal mn(ii ) binding site in the co - substituted 
 w132f oxdc variant ( figure 4 ) , the 40 k epr 
 spectrum showed evidence for a pentacoordinated mn(ii ) species 
 exhibiting the telltale sharp peaks at the low - field side and a pake 
 pattern at the near high - field side of the spectrum . as in our earlier 
 studies of wt oxdc , we were able to simulate the observed transitions 
 by assuming that only two mn sites were present ( figure 6 ) . hence , assuming a single mn(ii ) species having a negative d value of 1950 mhz and a large gaussian distribution 
 of e values centered around 400 mhz with a half - width 
 of 1200 mhz gave a reasonable simulation of the low - field side of 
 the spectrum even though the broad feature on the high - field side 
 could only be partially reproduced .
an improved simulation could be 
 obtained using a much broader non - gaussian spectral distribution of d and e in which d and e ranged between 1600 and 2100 mhz and between 
 100 mhz and the maximum value of |d|/3 , respectively 
 ( figure s10 in supporting information ) . 

both simulations , however , provide clear experimental evidence for 
 a significant increase in the magnitude of d for 
 one of the mn(ii ) sites in the w132f oxdc variant when compared to 
 wt enzyme .
this spectroscopic behavior is therefore consistent with 
 the hypothesis that removal of the glu-101/trp-132 hydrogen bond would 
 impact the electronic structure of the mn(ii ) ion bound in the n - terminal 
 domain .
high - field ( 326.4 ghz ) epr spectra of the mn - containing w132f oxdc 
 variant taken at 3 k ( black ) and 40 k ( blue ) together with spectral 
 simulations .
note that the central part of the spectrum at 40 k together 
 with the corresponding spectral simulation of site i is removed for 
 clarity .
the enzyme ( 40 mg / ml ) was dissolved in 50 mm tris buffer 
 containing 500 mm nacl , ph 8.5 .
lines from spectral simulations correspond 
 to a single mn(ii ) species ( site i ) with small zfs at 3 k ( red ) and 
 40 k ( blue ) and a single mn(ii ) species ( site ii ) with very large 
 zfs at 40 k ( cyan ) .
the sum of site i and site ii simulations at 40 
 k is also shown ( magenta ) .
we note that site ii does not contribute 
 significantly to the spectral range visible .
unedited spectra and 
 full sets of simulation parameters are provided elsewhere ( figures 
 s8s10 in supporting information ) .
the ability 
 of dft and wave - function - based methodologies ( widely and successfully 
 used to model mononuclear inorganic complexes ) to compute zfs values for a series of mn(ii ) transition metal complexes 
 has been evaluated .
perhaps , unsurprisingly , 
 these studies revealed that the computed zfs values are highly dependent 
 on molecular geometry .
the availability of good structural and experimental 
 zfs data for wt oxdc offered the opportunity to examine whether qm / mm 
 methods for obtaining optimized active site geometries might yield 
 structures that could ( at the very least ) be used to compute qualitatively 
 correct estimates of the zfs parameters for protein - bound mn(ii ) ions . 

thus , an initial structure for performing qm / mm geometry optimizations 
 was generated from classical md simulations of wt oxdc solvated in 
 water at physiological conditions .
although two x - ray structures have 
 been reported for the enzyme , we used the one containing a pentacoordinate 
 mn(ii ) ion in the c - terminal domain ( pdb i d : 1uw8 ) to obtain the initial 
 set of atomic coordinates for all subsequent calculations on wt oxdc 
 ( figure 1 ) .
after qm / mm optimization of the 
 metal centers in the initial enzyme , a set of cluster models were 
 defined in which only oh / h2o molecules in the active site 
 cavity and the side chains of all the residues in the first coordination 
 sphere around the metal were included ( figure 7 ) .
cluster models taken 
 from the dft / mm - optimized structure of wt 
 oxdc that were used to calculate the zfs values of the bound mn(ii ) 
 ions in the ( a ) n - terminal and ( b  d ) c - terminal domains ( see 
 text for details ) .
color scheme : c , cyan ; h , white ; n , blue ; oxygen , 
 red ; mn , tan .
these models were then 
 used in calculations of the zfs parameters 
 that employed the hybrid b3lyp functional 
 in conjunction with triple- ( def2-tzvp ) valence basis sets . 

remarkably , excellent agreement was obtained in the prediction of 
 both d and e / d values 
 when qm / mm - optimized geometries were used to obtain the atomic coordinates 
 for these models , showing the importance of the surrounding residues 
 in modulating the geometric features of the oxdc active sites ( table 1 and table s2 in supporting 
 information ) .
for example , the d value of 
 1170 mhz computed for the n - terminal mn(ii ) cluster model 
 ( figure 7a ) was similar in both sign and absolute 
 magnitude to that observed for site i ( d = 1350 
 mhz , table 1 ) .
computing good estimates for 
 the d value associated with the c - terminal mn(ii ) 
 center required more effort .
for example , an initial calculation on 
 a cluster in which water was used as the fifth ligand ( figure 7b ) gave a d value of 4110 
 mhz from b3lyp / def2-tzvp calculations , which is considerably smaller 
 than the observed |d| value of 10430 mhz ( table 1 ) .
closer examination of the x - ray crystal structure , 
 however , showed that the mn 
o distance in the c - terminal mn(ii ) 
 site was only 2.15  , whereas both mn  o distances for 
 the metal center in the n - terminal domain were significantly longer 
 ( 2.31 and 2.33  ) .
this observation thereby raised the possibility 
 that the fifth ligand in the c - terminal site was a hydroxide ion rather 
 than water . as a result , and
especially given that the crystal structure 
 ( 1uw8 ) had been 
 obtained at ph 8.5 , we examined the effects of modeling the fifth 
 ligand as a hydroxide ion .
we therefore reoptimized the wt oxdc following 
 the protocol previously outlined , except that hydroxide was bound 
 to the metal in the c - terminal binding site , and created another cluster 
 model of this mn(ii ) center ( figure 7c ) .
b3lyp / def2-tzvp 
 calculations using this third model gave a computed d value of 4560 mhz , which was a slight , albeit small , improvement . 

we noticed , however , that additional water molecules entered the c - terminal 
 mn(ii ) binding site during the initial set of classical md simulations 
 used in equilibrating the system .
one of these waters formed a hydrogen 
 bond to the mn(ii)-bound hydroxide ligand after dft / mm optimization . 

the b3lyp / def2-tzvp calculations were therefore repeated for a new 
 cluster model containing this water molecule ( figure 7d ) , with the result that a d value of 7130 
 mhz was obtained .
this is in much better agreement with the experimentally 
 observed value for the site ii mn(ii ) species .
we also note that the 
 calculated e / d ratio for site i 
 was similar to that obtained by analyzing the high - field epr spectra , 
 although any comparison of theoretical and our experimental e / d values is devalued by the broad distribution 
 of e values needed in the spectral simulations . despite 
 this complication
, our dft - based computational strategy again supports 
 the zfs assignments of tabares et al . as an additional validation of our strategy for obtaining accurate 
 geometries for mn(ii ) binding sites in proteins that permit practically 
 useful theoretical estimates of their corresponding zfs values , we 
 constructed an optimized model of the mn(ii)-containing w132f oxdc 
 variant based on the 2.1  resolution x - ray crystal structure 
 of the co(ii)-substituted enzyme .
after  replacing  
 the co(ii ) ions with mn(ii ) , our standard dft / mm procedure was used 
 to optimize the resulting structure .
as discussed above , the n - terminal 
 mn(ii ) coordination environment in the initial configuration exhibited 
 some unexpected features , including ( i ) rotation of the his-97 imidazole 
 ring by about 180 relative to its orientation in the x - ray crystal 
 structure of wt oxdc , and ( ii ) almost identical mn 
n 
 ( 2.45  ) and mn  c ( 2.47  ) distances for the 
 interaction of the metal with his-97 ( figure 5 ) .
in contrast , the final computational model of the mn(ii)-containing 
 w132f oxdc variant obtained using our standard protocol ( equilibration 
 using classical md simulation followed by dft / mm optimization ) did 
 not contain these unexpected structural features ( figure 8) . thus , the his-97 imidazole ring underwent a rotation 
 of about 70 with respect to its orientation in the initial structure , 
 and the mn  n ( 2.24  ) and mn  c ( 3.21 
  ) distances became appreciably different .
most importantly , 
 however , b3lyp / def2-tzvp calculations on a cluster model of the n - terminal 
 mn(ii ) site gave a d value of 2100 mhz in 
 very good agreement with experiment ( d = 1950 
 mhz ) ( table 1 ) .
superimposition of the 
 n - terminal mn(ii ) binding sites present 
 in the x - ray crystal structure of the co - containing w132f oxdc variant 
 ( green ) and the dft / mm - optimized structure of the mn(ii)-containing 
 w132f oxdc variant ( gray ) .
only the residues in the first coordination 
 sphere around the metal are depicted as sticks .
we also assessed 
 the ability of our computational protocol to be employed in 
 a predictive manner by calculating the mn(ii ) zfs values 
 for the n - terminal metal center in a model of the w132f oxdc variant 
 prepared by in silico mutation .
hence , the x - ray crystal structure 
 of wt oxdc ( 1uw8 ) was computationally modified 
 so that trp-132 was replaced by a phenylalanine residue .
the 
 resulting model was then equilibrated and optimized using our standard 
 protocol to give a final structure in which the hydrogen bond between 
 the glu-101 side chain , and the closest mn(ii)-bound water became 
 slightly shorter as a consequence of removing the interaction between 
 glu-101/trp-132 ( table 2 ) .
changes in other 
 metal  ligand bond distances were also observed together with 
 slight changes in the locations of the side chains of some nearby 
 residues .
once again , b3lyp / def2-tzvp calculations were performed 
 on a cluster model of the n - terminal mn(ii ) site corresponding to 
 that used for calculations on wt oxdc ( figure 7a ) .
the computed d value of 1350 mhz ( table 1 ) shows that this optimized model structure captures 
 the increased magnitude of d that is experimentally 
 observed on removal of the glu-101/trp-132 hydrogen bond . on the other 
 hand , the absolute value is smaller than that computed from a model 
 based on the x - ray crystal structure of the co(ii)-containing w132f 
 oxdc variant ( table 1 ) . given that superimposition 
 of the two cluster models used to compute the zfs parameters for the 
 n - terminal mn(ii ) site in the w132f oxdc variant showed all metal 
ligand 
 distances to be similar in both structures , the difference in the 
 calculated d values likely arises from altered orientations 
 of the imidazole rings of his-97 and ( to a smaller extent ) his-95 
 ( figure 9 ) . taken overall , these results suggest 
 that the computational strategy described in this paper yields reliable 
 structural information and , as a consequence , very good estimates 
 of zfs values for protein - bound mn(ii ) centers . importantly , our approach 
 opens the possibility of computing fine structure parameters in biomolecular 
 systems for which only lower - resolution crystal structures are available .
qm / mm - optimized 
 structure with the 
 bound hydroxide in the c - terminal mn(ii ) center forming a hydrogen 
 bond to a water molecule .
this structure was obtained from the x - ray 
 crystal structure of wt oxdc ( 1uw8 ) ( figure 7a , d ) .
qm / mm - optimized structure with 
 water 
 bound to the c - terminal mn(ii ) center based on the x - ray crystal structure 
 ( 1uw8 ) ( figure 7b ) .
qm / mm - optimized structure with hydroxide 
 bound to the c - terminal mn(ii ) center based on the x - ray crystal structure 
 ( 1uw8 ) ( figure 7c ) .
qm / mm - optimized structure obtained 
 from the x - ray crystal structure of the co(ii)-containing w132f oxdc 
 variant .
qm / mm - optimized 
 structure obtained 
 from in silico substitution of the x - ray crystal structure of wt oxdc 
 ( 1uw8 ) .
superimposition of the cluster models used to 
 calculate the zfs 
 parameters of the mn(ii ) n - terminal binding site in the w132f oxdc 
 variant .
color scheme : green , initial structure based on the x - ray 
 crystal structure of the co(ii)-containing variant ; orange , initial 
 structure derived from the x - ray crystal structure of wt enzyme ( in 
 silico substitution ) .
in 
 addition to resolving the question of mn(ii ) assignments for 
 oxdc , this study provides further evidence for the utility of advanced 
 quantum mechanical calculations in probing the electronic structure 
 of protein - bound metal centers . to date
, there have been few such 
 calculations , in part because previous efforts to compute zfs parameters 
 on a series of mn(ii)-containing complexes gave large differences 
 between theoretically predicted and experimentally measured values . 

these discrepancies can be attributed to the high sensitivity of the 
 zfs parameters to the geometrical features of the structures used 
 for the calculations .
it is also true that the requirement that zfs 
 parameters reflect only small energy differences places considerable 
 demands on the computational methods used to obtain them , and only 
 a qualitative agreement giving rise to correct trends can be expected 
 in general . on this point , we note that the overestimated d values found in efforts to compute the zfs parameters 
 for the mn(ii ) ions bound to enolase were 
 attributed both to the high sensitivity of the calculated values to 
 the molecular geometries employed and the use of dft calculations 
 instead of correlated ab initio methods .
however , some of the limitations 
 associated with the use of dft methodologies might be overcome by 
 employing the more recently developed coupled - perturbed spin 
orbit 
 coupling ( cp - soc ) formalism ( as used here ) instead of the pederson 
 and khanna ( pk ) approach used in the previous work on enolase - bound 
 mn(ii ) ions , provided that the direct spin  spin interaction 
 is properly included in the treatment . in particular
, systematic investigation 
 of the ability of coupled - perturbed dft methodologies to estimate 
 zfs parameters in mn(ii ) transition metal complexes also showed that reliable results could be obtained using 
 a hybrid dft functional ( b3lyp ) if accurate geometries were employed .
the theoretical results reported herein , however , demonstrate that 
 the use of a strategy based on computation of zfs parameters at the 
 dft theory level for cluster models obtained from qm / mm - optimized 
 structures has some value , especially if high - quality x - ray structures 
 are available for the protein , or site - specific variant , of interest . 

indeed , the reorientation of the his-97 side chain observed upon the 
 application of our computational protocol was likely an essential 
 element in obtaining good agreement between the calculated and experimentally 
 observed zfs parameters . our results also show that qm / mm - optimized 
 models of variant structures can also yield qualitatively correct 
 changes in computed zfs values , especially when the site - specific 
 mutation does not introduce large structural changes in the metal 
 ion environment .
presumably this reflects the ability of carefully 
 equilibrated qm / mm structures to capture the critical metal 
ligand 
 interactions , such as metal  ligand distances and coordination 
 geometries , needed to obtain good estimates of zfs parameters .
although 
 additional systematic computational investigations on the problem 
 of computing zfs values are clearly needed , our findings set the scene 
 for theoretical studies aimed at delineating the effect of active 
 site modifications on the electronic structure of the metal centers 
 in oxdc and , perhaps , other mn - dependent enzymes .
unless otherwise stated , all chemicals were purchased in the highest 
 purity from sigma ( st .
nickel - nitrilotriacetic acid agarose ( ni - nta ) was obtained 
 from qiagen ( germantown , md ) .
protein concentrations were determined 
 using the bradford assay ( pierce , rockford , il ) .
( coralville , ia ) , and dna sequencing was performed by the core 
 facility in the interdisciplinary center for biotechnology research 
 at the university of florida .
icp - ms determinations of metal content 
 were performed at the university of georgia center for applied isotope 
 studies chemical analysis laboratory ( athens , ga ) . the gene encoding 
 the w132f oxdc variant was constructed by overlap extension ( see supporting information ) .
the recombinant his6-tagged mn(ii)-substituted w132f 
 oxdc variant was expressed and purified following published procedures , except that expression was induced in the presence 
 of 5 mm mncl2 after heat shocking the bacteria for 12 min 
 at 42 c with constant agitation . after the addition of iptg , 
 the cultures were incubated at 37 c for 4 h before being harvested 
 by centrifugation at 2000 g for 20 min ( 4 c ) . 

after sonication in lysis buffer , the resulting supernatant was centrifuged 
 at 20  000 g for 20 min ( 4 c ) before the 
 enzyme was purified from the cleared lysate by metal affinity chromatography 
 on a ni - nta column .
the eluted protein was then subjected to dialysis 
 at 4 c to give a solution in 50 mm tris buffer containing 500 
 mm nacl , ph 8.5 .
samples used for epr analysis were incubated with 
 chelex resin before being concentrated to 27 mg / ml using an amicon 
 centriprep ym-30 filter unit from millipore ( billerica , ma ) .
the catalytic 
 properties of the his6-tagged mn(ii)-containing w132f oxdc 
 variant were determined by measuring formate production using an end - point 
 assay as described elsewhere .
measurements 
 were made at specific substrate and enzyme concentrations in triplicate , 
 and the data were analyzed to obtain the values of v and v / k by standard computer - based 
 methods .
his6-tagged co(ii)-containing 
 w132f oxdc was expressed as described previously with the addition 
 of 2 mm cocl2 after heat shocking the cells at 42 c 
 for 18 min .
cells were lysed using a microfluidizer , and the mixture 
 was clarified by centrifugation at 38  000 g for 30 min .
the eluate was dialyzed overnight in the storage buffer 
 ( 50 mm tris - hcl , ph 8.5 , and 500 m nacl ) , and the protein was then 
 further purified on a 320 ml sephacryl s-100 gel filtration column .
purified co(ii)-containing w132f oxdc was concentrated to 5 mg / ml 
 in 100 mm tris - hcl , ph 8.5 , containing 500 mm nacl .
crystals were 
 obtained by the vapor diffusion method with hanging drop geometry 
 by mixing protein ( 2 l ) and well solution ( 2 l ) .
the 
 well solution contained 100 mm tris - hcl , ph 8.5 , 2 m nacl and 10% 
 peg 6000 . crystals , which were rectangular prisms ( 0.3  0.2 
  0.2 mm ) , appeared in approximately 2 months at 17 c and 
 were cryo - protected in well solution containing 25% glycerol and flash 
 frozen in liquid nitrogen .
the co(ii)-substituted w132f variant crystallized 
 in space group r32 with unit cell dimensions a = b = 156.973  and c = 330.473  .
data were collected to 1.9  resolution at 
 the national synchrotron light source ( nsls ) , brookhaven national 
 laboratory ( upton , ny ) , at beamline x25 equipped with a pilatus 6 m 
 detector ( table 3 ) .
phasing was achieved by 
 molecular replacement ( phaser - mr in the 
 phenix software suite ) using the model 
 of formate - containing wt oxdc ( pdb 1j58 ) after 
 removal of all ligands .
the structure was refined using alternating 
 rounds of manual rebuilding in coot followed 
 by minimization in phenix to give a final model that contained residues 
 6382 with 4 molecules in the asymmetric unit , 1228 waters , 
 and 8 co(ii ) ions .
the model was refined to 2.1  resolution 
 with a rwork and rfree of 19.6 and 22.7% , respectively ( table 3 ) .
the ramachandran plot shows that 97.6% of residues fall 
 in the most favored regions with 2.3% in the allowed regions .
the 
 maximum likelihood coordinate error is 0.23  , consistent with 
 accurate coordinates .
coordinates and structure factors for the co(ii)-containing 
 w132f variant have been deposited in the pdb with accession code 4met .
ihkli|/hkli|ihkl , i | , where ihkl is the mean intensity of the multiple ihkl , i observations for symmetry - related 
 reflections .
high - field 
 epr spectra at 324.6 ghz were recorded at the national high magnetic 
 field laboratory ( nhmfl ) , using a homodyne transmission - mode spectrometer 
 with a nonresonant probe and a 17 t superconducting magnet .
wt oxdc ( 27 mg / ml ; 1.4 mn / monomer ) or the w132f 
 oxdc variant ( 40 mg / ml ; 1.6 mn / monomer ) was dialyzed into 50 mm tris , 
 ph 8.5 , containing 500 mm nacl .
approximately 200 l of this 
 solution was placed into a teflon cup with 7.2 mm i.d . and placed 
 into the fir beam of the spectrometer at 4 k , as measured using a 
 built - in carbon - glass temperature sensor in the cryostat .
a cernox 
 sensor was used to map out the temperature scale at the sample position 
 to correct for any temperature gradient in the cryostat .
spectra were 
 taken with a modulation amplitude of approximately 27 g at a modulation 
 frequency of 30 khz using a phase - sensitive lock - in detector with 
 the time constant set to 300 ms .
the spectra were simulated based on the usual spin 
 hamiltonian ( shown below ) using the easyspin toolbox for matlab .
md simulations of the wild - type form 
 of oxdc were based on the 1uw8 crystal structure 
 of the enzyme .
two different procedures were employed to build the 
 w132f oxdc variant : ( i ) in silico mutation , manually substituting 
 trp-132 by phe in the 1uw8 x - ray structure , and ( ii ) replacement of co with mn 
 using the co - substituted w132f oxdc mutant as template . in every case , 

the initial structure was immersed in a periodic box of approximately 
 77  118  87  containing about 22  500 
 water molecules and neutralized with na counterions .
the 
 box dimensions were chosen to achieve a minimum distance of 30  
 between two periodically replicated images of the protein .
the all - atom 
 amber03 force field was used to model 
 protein residues and ions , whereas the tip3p model was employed for water molecules .
all potentially charged 
 amino acids , including the c- and n - termini , were considered to be 
 in their default protonation states at physiological ph ( i.e. , charged ) . 

electrostatic interactions were taken into account using the particle 
 mesh ewald algorithm with a real space 
 cutoff of 10  .
the same cutoff was employed for the treatment 
 of the van der waals interactions .
constant temperature 
 ( 300 k ) and pressure ( 1 atm ) were achieved by coupling the systems 
 to a langevin thermostat and a nos 
systems were first 
 minimized using a conjugate gradient algorithm and then heated to 
 300 k in 600 ps while keeping positional restraints on protein heavy 
 atoms . for each system , an initial run of 5.4 ns in the npt ensemble , 
 slowly removing the restraints , was followed by a subsequent 20 ns 
 simulation in the canonical ( nvt ) ensemble in order to provide starting 
 configurations for the subsequent qm / mm optimizations .
qm / mm 
 geometry optimizations 
 were performed starting from equilibrated configurations taken from 
 the aforementioned classical runs .
the quantum mechanical / molecular 
 mechanical ( qm / mm ) implementation employed combines the use of the 
 qm program quickstep and the mm driver 
 fist , both part of the cp2k package ( freely available at http://cp2k.berlios.de , released under gpl license ) . in this code
, the general qm / mm scheme 
 is based on a real - space multigrid technique to compute the electrostatic 
 coupling between both qm and mm regions . in all the optimizations , a quantum region consisting of the mn 
 center together with
the residues at a distance shorter than 6  
 from it ( including only the side chains up to the c atom ) was treated at the density functional theory level , whereas 
 the remaining part of the system , including water molecules and counterions , 
 was modeled at the classical level using the amber force field to 
 take explicitly into account the steric and electrostatic effects 
 of the surroundings .
the valence of the terminal qm atoms was saturated 
 by the addition of capping hydrogen atoms .
a dual basis set , gaussian 
 and plane - wave formalism , was employed to compute the interaction 
 energy within the qm subsystem .
a molecularly optimized double- 
 valence basis set augmented with polarization functions ( m - dzvp ) was 
 used to describe the wave function , while 
 an auxiliary plane - wave basis set expanded up to a density cutoff 
 of 360 ry was utilized to converge the electron density in conjunction 
 with goedecker  teter 
exchange and correlation energies 
 were computed within the generalized gradient approximation by using 
 the blyp functional .
the calculation of 
 the soc and ss parts of the zfs parameters was performed with the 
 orca electronic structure package .
we 
 performed these calculations on cluster models , in which only the 
 oh / h2o molecules in the active site cavity and the side 
 chains of all the residues in the first coordination sphere around 
 the metal were included .
functional 
 in conjunction with triple- ( def2-tzvp ) valence basis sets , taking advantage of the rijcosx approximation to speed up the calculations . in all cases , 

the coupled - perturbed 
 soc approach was used to evaluate the dsoc contribution , whereas the dss contribution 
 was obtained on the basis of the spin - unrestricted natural orbital 
 determinant .
scalar relativistic effects 
 were taken into account by means of the van wllen s 
 model potential approximation to the zora equations .
 oxalate 
 decarboxylase ( oxdc ) catalyzes the mn - dependent conversion 
 of the oxalate monoanion into co2 and formate . 
 epr - based 
 strategies for investigating the catalytic mechanism of decarboxylation 
 are complicated by the difficulty of assigning the signals associated 
 with the two mn(ii ) centers located in the n- and c - terminal cupin 
 domains of the enzyme . 
 we now report a mutational strategy that has 
 established the assignment of epr fine structure parameters to each 
 of these mn(ii ) centers at ph 8.5 . 
 these experimental findings are 
 also used to assess the performance of a multistep strategy for calculating 
 the zero - field splitting parameters of protein - bound mn(ii ) ions . 
 despite the known sensitivity of calculated d and e values to the computational approach , 
 we demonstrate that 
 good estimates of these parameters can be obtained using cluster models 
 taken from carefully optimized dft / mm structures . 
 overall , our results 
 provide new insights into the strengths and limitations of theoretical 
 methods for understanding electronic properties of protein - bound mn(ii ) 
 ions , thereby setting the stage for future epr studies on the electronic 
 properties of the mn(ii ) centers in oxdc and site - specific variants . 
Introduction
Results
and Discussion
Conclusions
Experimental Section