# Supplementary Material (ESI) for Chemical Communications
# This journal is © The Royal Society of Chemistry 2004
Supplementary Information for
The influence of solvation on short strong hydrogen bonds: A density functional theory study
of the Asp-His interaction in subtilisins.
Birgit Schiøtt, Department of Chemistry, University of Aarhus, DK-8000, Århus, Denmark
Computational Methods.
The model used in the study is taken from the high resolution structure of Bascillus lentus Subtilisin (pdbcode 1GCI) by extracting the side-chain coordinates of His64 and Asp32 and of water1059 form the pdbfile.1 Only the hydrogen atom between Asp32 and His64 was localized in the X-ray diffraction experiment,1
all remaining hydrogen atoms are added using the Quanta2000 modelling environment. 2 The resulting model
consists of an aspartate ion, a methyl imidazolium and a water molecule. All quantum chemical calculations
in this study are carried out by using the Gaussian98 suite of programs.3 Initial structures are computed at the
HF/6-31+G(d) level of theory and these structures are subsequently used as starting geometries for all
production calculations which use the B3LYP hybrid method and includes continuum solvation.
The initial structure was found by finding the preferred orientation of the hydrogen atoms of the water
molecule at the HF/6-31+G(d) level of theory and keeping the remaining system fixed. Then, starting
structures were generated allowing the full system to relax at the same level of theory. Structures are found
where the hydrogen of interest is localized on His64(N1) as well as on Asp32(O2), Scheme 2 in the paper.
The transition state for hydrogen transfer is localized using the Synchronous Transit –guided Quasi Newton4
technique. It should be noted, that when leaving the water molecule out from the model at the HF/6-31+G(d)
level of theory, the N-side isomer does not represent a minimum of the potential energy surface – only the
structure of the O-side isomer can be optimized without inclusion of the explicit water molecule.
All numbers and results reported in the paper are calculated at the B3LYP(COSMO)/6-31++G(d,pd) level
of theory based on the following arguments. Experimental charge density studies of model complexes of the
charge relay system between His and Asp have appeared recently.5 These studies shows that the hydrogen
atom of interest needs multipole expansions to a quadrupole level to model the electron density in the
hydrogen bonding region.5a,b Therefore we choose for all production calculations in this study to use a basis
set that includes d-orbitals on hydrogen atoms and also has diffuse orbitals on all atoms, consequently the 631++G(d,pd) basis set is used.6 For intermolecular hydrogen-bonded systems, it has been shown that DFT
with the B3LYP hybrid functional7 represents a very good method for structural optimizations,8 and we have
previously successfully used this method to study the short strong hydrogen bond in benzoylacetone. 5c,d In
the gas phase it is not possible to identify the N-side isomer at the B3LYP/6-31++G(d,pd) level of theory,
neither with nor without the explicit water molecule. In either case, the potential energy curve only has one
minimum corresponding to the O-side isomer, presumably because charged species are not stable in vacuum.
Therefore, continuum solvation was included in the calculations. Continuum solvation effects are modeled
using the implementation of the conductor-like screening solvation model9 (COSMO) in Gaussian98.10 The
medium is chosen to be diethylether with a dielectric constant  = 4.33511 to model the hydrophobic interior
of an enzyme. Exclusion of the water molecule using the B3LYP(COSMO) mothodology also failed to give
the double-well potential curve. All stationary points are characterized by frequency analyses, and the
number of imaginary frequencies is zero for all minima and exactly one for all transition states found. For
the saddle points, the imaginary frequency produced is analyzed by the the Molden program12 to confirm that
the located TS in fact corresponds to the hydrogen transfer, and not e.g. to a methyl rotation. Fully
optimized geometries for the structures 1, 2 and 3, calculated at the B3LYP(COSMO)/6-31++G(d,pd) level
of theory are listed below.
# Supplementary Material (ESI) for Chemical Communications
# This journal is © The Royal Society of Chemistry 2004
Acknowledgement:
The Danish Natural Science Research Council is gratefully acknowledged for a Steno-grant and the Novo
Nordisk Foundation for a research grant. Computations were made possible through allocation of time at
DCSC, Aarhus, using the Origin 2000 machine. Finally, fruitful discussions with to Prof. T. C. Bruice are
greatly appreciated.
N-side (1)
N 0.996243
C 1.156816
N 2.472676
C 3.173921
C 2.240669
C 2.408403
O -1.138738
C -2.356542
O -2.735538
C -3.407412
O -1.541475
H -3.235133
H -4.413071
H -3.317075
H 0.004136
H 3.468670
H 1.951314
H 1.936450
H 0.349420
H 2.877223
H 4.251163
H -1.988829
H -2.135827
0.024436
0.025280
0.015888
0.007969
0.013687
0.009999
0.034762
-0.005157
-0.049868
-0.008243
-0.093843
0.826060
0.055526
-0.928978
0.026703
0.000577
0.895748
-0.869179
0.033026
0.012749
-0.000304
-0.072272
0.348461
0.078241
1.399051
1.662073
0.469425
-0.532903
-2.014769
-1.286693
-0.893140
0.304686
-1.993613
2.667481
-2.678950
-1.577852
-2.578514
-0.450546
-2.273953
-2.465700
-2.463212
2.125454
2.592480
0.435572
1.778116
3.286810
O-side (2)
C -1.379603
N -2.730971
C -3.225469
C -2.128290
N -0.983027
C -2.071544
O 1.378682
C 2.489822
C 3.735612
O 2.530950
O 1.092957
H 3.757223
H 4.622955
1.162446
1.230271
-0.059370
-0.885059
-0.103175
-2.378291
-1.223164
-0.516189
-1.365932
0.715373
3.044104
-1.980888
-0.735752
-0.042233
-0.021275
0.011764
0.009668
-0.024609
0.037646
-0.042208
-0.006880
-0.003949
0.025466
0.065554
0.900649
-0.039276
# Supplementary Material (ESI) for Chemical Communications
# This journal is © The Royal Society of Chemistry 2004
H
H
H
H
H
H
H
H
H
H
3.725626
0.495904
-3.078378
-1.527086
-1.562271
-0.732350
-3.277469
-4.282705
1.624996
1.687664
-2.047410
-0.668938
-2.801198
-2.739147
-2.773445
2.029139
2.083057
-0.271155
2.221456
3.758464
-0.858677
-0.033648
0.065924
0.916270
-0.847296
-0.068358
-0.030274
0.034483
0.052151
-0.197341
TS (3)
N -0.956318
C -1.354320
N -2.699496
C -3.178650
C -2.078910
C -1.982593
O 1.291447
C 2.422594
O 2.560910
C 3.646979
O 1.090830
H 3.669549
H 4.560301
H 3.590976
H 0.197198
H -2.979553
H -1.452908
H -1.438222
H -0.699117
H -3.261908
H -4.233067
H 1.640321
H 1.601035
-0.080887
1.186792
1.221200
-0.075513
-0.893293
-2.382031
-1.129209
-0.508357
0.732195
-1.404135
2.970565
-1.964354
-0.817426
-2.136414
-0.498069
-2.826801
-2.765656
-2.721009
2.050464
2.065102
-0.299017
2.148510
3.653296
0.021954
0.048271
0.033139
-0.004478
-0.011698
-0.048533
0.039239
0.000872
-0.050289
0.027911
-0.091199
0.967524
-0.066840
-0.782160
0.027638
-0.068253
0.828470
-0.934992
0.074080
0.044435
-0.023526
-0.071112
0.363007
1
2
3
4
5
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# Supplementary Material (ESI) for Chemical Communications
# This journal is © The Royal Society of Chemistry 2004
8
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