Download IJCA 50A(09-10) 1457-1462

Indian Journal of Chemistry
Vol. 50A, Sept-Oct 2011, pp. 1457-1462
Density functional theory calculations on biological S-transfer: Insight into the
mechanism of rhodanese
Subal Dey & Abhishek Dey*
Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700 032, India
Email: [email protected]
Received 8 June 2011; revised and accepted 10 August 2011
Biological sulfur transfer (S-transfer) is a key step in the synthesis of metabolites, CN- detoxification and assembly of
iron-sulfur clusters. Computational results addressing the thermodynamics of the S-transfer reactions from thiosulfate
(natural S-donor) to HCN and thiol are presented. These calculations indicate that S-transfer from thiosulfate to HCN and
thiol is possible only in the anionic forms of these species. However these species have pKa values significantly higher than
physiological pH value (i.e., they are protonated in physiological pH and incapable of S-transfer). In the rhodanese active
site, basic residues are present to deprotonate the catalytic cysteine group which accepts the S-atom from thiosulfate. The
resultant perthiol species transfer S-atom to CN- in a synchronous S-atom and H+ transfer step facilitated by the two arginine
residues present in the rhodanese active site. Based on these calculations, a mechanism is proposed for the rhodanese
catalyzed CN- detoxification pathway.
Keywords: Density functional calculations, S-transfer reactions, Cyanide detoxification, Rhodanese
The presence of sulfur in biological cofactors has
been established since the early 20th century.1 Various
vitamins, amino acids, iron-sulfur clusters, lipoic acid,
biotin and thiouridine contain sulfur atom as
functional motifs of these biologically important
compounds.2 Photosynthetic sulfur bacteria use
the sulfur compounds as a source of electrons for
reductive carbon dioxide fixation during anoxic
autotropic growth, while chemolithoautotropic sulfur
bacteria uses the electron derived from oxidation of
inorganic sulfur species both in carbon dioxide
fixation and as respiratory electron donors.3,4 In the
last decade great strides have been made towards
understanding the biosynthesis of these processes.
It has been established that the cystein desulfurase
and the rhodanese homology domains are essential
for catalyzing the insertion of the sulfur atoms into
bimolecules.2,5 Remarkably, all the enzymes use
perthiols as an intermediate for the sulfur transfer,
when perthiols, as a chemical entity, is quite
uncommon in synthetic preparation.6 Persulfides, on
the other hand, are well characterized.7-9
Rhodanese an ubiquitous structural module
occurring in three major evolutionary phyla, is
the enzyme that can catalyse the conversion of
thiosulfate to thiocyanide in the presence of cyanide,
i.e., thiosulfate:cyanide sulfurtransferase(TST).10-12
Rhodanese from bovine13 and Azatobacter vinelandii
(RhdA)14 is crystallographic characterized. It consists
of two equally-sized globular domains:13 the active
C-terminal domain which contains the active site
loop hosting six amino acid residues with one
cysteine residue at the first position and the
catalytically inactive N-terminal domain where
the Cys residue is replaced by an Asp residue.13,15-17
The peculiarity of rhodanese resides in two conserved
patterns of amino acid sequences at the N-terminal
region ([F/Y]-X3-H-[L/I/V]-P-G-A-X2-[L/I/V]) and at
the C-terminal end of the protein ([A/V]-X2-[F/Y][D/E/A/P]-G-[G/S/A]-[W/F]-X-E-[F/Y/W]).18
The sulfur transfer catalysis at the rhodanese active
site occurs via a proposed double displacement
mechanism involving the formation of a transient
cysteine persulfide intermediate,2 in which the
transferring sulfur is bound to the catalytic Cys
residue (Fig. 1). This sulfane sulfur (S0) is proposed to
be nucleophilic in nature and easy to deliver to
cyanide to form thiocyanide in the second step of the
reaction.19-22 The catalytic cysteine sulfur is H-bonded
to basic arginine residues which are also proposed to
be mechanistically important. It may be assumed as
the cyanide detoxification pathway within the cell and
may appear as the major contributing component of
the recovery mechanism for the native architecture of
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INDIAN J CHEM, SEC A, SEPT-OCT 2011
Table 1—Calculated solvation enthalpies
Species
Solvation enthalpy (kcal/mol)
-
CH3S
CH3SH
HSCN
CNCH3SSH
SCNCH3SSHCN
S2O32SO32-
Fig. 1—Active site of bovine rhodanese from (pdb id: 2ORA).
The hydrogen bonding interaction with the catalytic Cys254
residue and Arg110 and Arg116 are indicated with dashed lines.
the iron-sulfur protein(s) as it can mobilize sulfur
for the formation or repair of iron-sulfur clusters.22,23
In spite of the importance of this reaction in biology,
little is known about the thermodynamics of these
reactions.
In this paper, the feasibility of the S-transfer
between several relevant species has been
investigated by density functional theory calculations
in the gas phase as well as using a solvation model.
These calculations provide insight into the possible
role and mechanism of rhodanese.
Computational Details
All calculations were performed in the HPC cluster
at IACS, Kolkata, using Gaussian 03 software
package. The geometries were optimized using
spin restricted formalism using both BP8624,25 and
B3LYP26-28 functional and a 6-311g* basis set.
All coordinates were allowed to optimize. Frequency
calculations at the end of geometry minimization
were done to ensure a stable minimum. The optimized
structures reported had no imaginary frequencies.
The total energies were calculated using 6-311+g*
basis set on all atoms. A polarized continuum model
(PCM) using water as solvent was used to model
solvation.29 Free energies differences (∆G) were
calculated by correcting the differences in electronic
energies (∆E) obtained using a 6-311+g* basis with
the entropy (S) and zero point energies (ZPE)
obtained during the frequency calculations using
6-311g* basis set. The results obtained using B3LYP
and BP86 functional were qualitatively the same so
only those obtained using BP86 is reported here.
-65.226
-0.32484
-8.45525
-79.2471
-2.03738
-61.6453
-60.513
-4.08202
-224.733
-276.194
Results and Discussion
Table 1 lists the calculated solvation energies for
the species relevant to this study. The results are
divided into two sections: Section A discusses the
thermodynamics of S-transfer from thiosulfate. In
this section thiosulfate is always assumed to be in its
dianionic form as the pKa of H2S2O3 is 0.6 and 1.72
(ref. 30). Thus, thiosulfate should exist in its dianionic
form in physiologically relevant pHs. In section B,
S-transfer from perthiolate species is considered. In
this section both the protonated and the deprotonated
forms are considered since perthiol has a pKa of
6.2 ± 0.1 and it can exist in both neutral as well as
anionic forms depending on the pH or the nature of
neighboring residue (i.e., basic residues, like Arg116
can deprotonate a perthiol.).31 Transfer of S-atom
along with a proton has also been considered as there
is a conserved basic group in the rhodanese active site
that can allow this synchronous shuttling.
(A) S-transfer from thiosulfate to
(i) Cyanide: S-transfer from thiosulfate to
cyanide
produces
thiocyanide
and
sulfite:
2−
−
2−
−
S2O3 + CN → SO3 + SCN . This reaction is
calculated to be thermodynamically favorable in the gas
phase (∆E = −4.514 kcal/mol, ∆G = −4.906 kcal/mol).
This reflects differences in the bond strengths
between the product and the reactant. In a PCM
model using water as solvent, this reaction is
calculated to be favorable (∆E = −18.504 kcal/mol,
∆G = −18.897 kcal/mol). The solvation of SO32− is
greater than S2O32− by 52 kcal/mol (Table 1), while
the solvation energy of SCN- is lower by 18 kcal/mol.
Thus, the greater solvation of the product SO32provides the driving force for the reaction in an
aqueous environment. There is minimal entropic
contribution to the calculated ∆G of this reaction.
DEY & DEY: DFT CALCULATIONS ON BIOLOGICAL S-TRANSFER REACTIONS
(ii) Hydrocyanic acid: The sulfur transfer from
thiosulfate to hydrocyanic acid will result in the
formation of hydrothiocyanic acid and sulfite:
HCN + S2O32− → HSCN + SO32−. The thermodynamic
values (∆E = 68.934 kcal/mol and ∆G = 66.86 kcal/mol)
indicate that the reaction is unfavorable. Here, proton
affinity (PA) plays the leading role. The gas phase
PA of CN− (−26.059 kcal/mol) is significantly greater
than the gas phase PA of SCN− (1.473 kcal/mol). This
makes the reaction thermodynamically unfavorable.
Upon introducing solvation correction, the ∆G is
lowered from +66.86 kcal/mol in the gas phase to
8.53 kcal/mol in water because of the larger solvation
energy of SO32- (product) relative to that of S2O32−
(reactant). However, the higher PA of HCN relative to
the PA of HSCN disfavors the S- transfer from
thiosulfate to hydrocyanic acid.
(iii) Methylthiolate: S-transfer from thiosulfate
to thiolate will yield sulfite and perthiolate:
CH3S− + S2O32− → CH3SS− + SO32−. The reaction
is calculated to be thermodynamically unfavorable
in the gas phase (∆E = 51.71 kcal/mol and
∆G = 50.51 kcal/mol). This is probably due to
weakening of the S−S bond in perthiolate species
relative to that in thiosulfate. However, the reaction is
calculated to be slightly thermodynamically favorable
after solvation correction (∆E = −0.661 kcal/mol
and ∆G = −1.86 kcal/mol). The solvation energy
of the reactant methylthiolate (−65.23 kcal/mol) is
~5 kcal/mol more than the solvation energy of
methylperthiolate (−60.51 kcal/mol). This will tend to
make the ∆G of the reaction unfavorable. However
the large solvation energy of SO32− overcomes this as
well as the gas phase endothermicity to enable the
reaction in the aqueous medium.
(iv) Methylthiol: Methylthiol on reacting with
thiosulfate will generate methylperthiol and sulfite:
CH3SH + S2O32− → CH3SSH + SO32−. From the
calculations we can conclude that this reaction is
thermodynamically unfavorable in the gas phase as
well as in the aqueous media. ∆E = 63.25 kcal/mol
and ∆G = 62.28 kcal/mol in the gas phase imply
that the S−S bond in perthiol is weaker than the
S−S bond in thiosulfate. Further, the gas phase
PA of methylthiol (−29.65 kcal/mol) is ~ 8 kcal/mol
higher than the gas phase PA of methylperthiol
(−21.83 kcal/mol). Both these factors make the
reaction thermodynamically unfavorable. Although
solvation correction favors this reaction (due to
the large solvation energy of SO32−), it does not
1459
overcome the gas phase endothermicity of this
reaction (∆G = 10.31 kcal/mol in water).
(B) (I) S− transfer from CH3SS− to
(i) Cyanide: Perthiolate to cyanide S-transfer will
result in the formation of thiocyanate and thiolate:
CH3SS− + CN− → CH3S− + SCN−. This reaction is
calculated to be favorable in the gas phase as well
as in water. In the gas phase, formation of stronger
C−S bond in SCN− and breaking of weak S−S bond
in perthiolate makes the reaction favorable
(∆E = −35.668 kcal/mol and ∆G = −34.967 kcal/mol).
Including solvation correction makes the reaction less
thermodynamically favorable (∆E = 21.977 kcal/mol
and ∆G = −21.276 kcal/mol). This is because the
calculated solvation energy of the reactant CN−
(−79.25 kcal/mol) is ~18 kcal/mol more negative than
the calculated solvation energy of the product SCN(−61.64 kcal/mol). Thus, in an aqueous medium the
reactant is stabilized relative to the product making
the ∆G more positive.
(ii) Hydrocyanic acid: Methyl perthiolate on
reaction with hydrocyanic acid results in the
formation of methyl thiolate and thiocyanic acid:
CH3SS− + HCN → CH3S− + HSCN. The reaction is
not favorable in the gas phase (∆E = 13.916 kcal/mol
and ∆G = 13.086 kcal/mol) because the proton
affinity of the reactant CN− (−26.68 kcal/mol) is
much greater than that of the product, SCN−
(1.47 kcal/mol). Though the solvation of the ions
make the ∆E or ∆G values less positive, the
reaction is still thermodynamically unfavorable
(∆E = 6.181 kcal/mol and ∆G = 5.351 kcal/mol). The
solvation energy of HCN and HSCN is contributing
only ~4 kcal/mol to the ∆G (solvation of HSCN is
greater). CH3S- has solvation energy only ~5 kcal/mol
more than that of CH3SS-. Both of these are not
sufficient to overcome the gas phase endothermicity.
(B) (II) S-transfer from CH3SSH to
(i) Cyanide: When sulfur atom is transferred
from methyl perthiol to cyanide anion it will
generate
methyl
thiol
and
thiocyanate:
−
−
CH3SSH + CN → CH3SH + SCN . This reaction is
exothermic in the gas phase (∆E = -50.42 kcal/mol
and ∆G = -64 kcal/mol). The major factor behind
driving this reaction is the formation of much stronger
C-S bond and breaking of the weak S-S bond. Also,
the gas phase PA values indicate that formation of
methyl thiol (-29.64 kcal/mol) from methyl perthiol
(-21.63 kcal/mol) is contributing a minor amount to
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INDIAN J CHEM, SEC A, SEPT-OCT 2011
the total energy. In an aqueous environment the
reaction becomes energetically less feasible (from
∆G = −29.795 kcal/mol to ∆G = −43.43 kcal/mol).
This +20 kcal/mol difference between the gas phase
and the solvent corrected ∆G can be accounted for
by the lower solvation energy of SCN- relative to the
solvation energy of CN−.
(ii) Hydrocyanic acid: Sulfur transfer from
methyl perthiol to hydrocyanic acid will
produce
methyl
thiol
and
hydrothiocyanic
acid: CH3SSH + HCN → CH3SH + HSCN. This
reaction is calculated to be almost thermoneutral
(∆E = 1.867 kcal/mol and ∆G = 0.811 kcal/mol). Upon
solvation, the reaction becomes slightly more favorable
(∆E = −1.63 kcal/mol and ∆G = −2.69 kcal/mol).
Slightly greater hydration energy of the products than
the reactants may account for this observation.
−
(B) (III) S and H+ transfer from CH3SSH to – CN
In addition to transfer of S atom from S donors,
synchronous transfer of both S and H+ group has
also been considered. When S and H+ groups are
transferred from methyl perthiol to cyanide, methyl
thiolate and hydrothiocyanic acid are the expected
products: CH3SSH + CN− → CH3S− + HSCN.
The calculations indicate that this reaction is
thermodynamically feasible. (∆E = −6.195 kcal/mol
and ∆G = −6.877 kcal/mol). SCN− has a lower
PA (1.47 kcal/mol) than that of CH3SS−
(−21.83 kcal/mol) which disfavors the reaction.
However, breaking of weaker S-S bond in CH3SSH
and formation of a strong C-S bond in HSCN drives
the reaction. Upon inclusion of solvation correction,
the reaction is calculated to be thermoneutral
(∆E = 1.327 kcal/mol and ∆G = 0.654 kcal/mol.).
The hydration energy of cyanide (−79.25 kcal/mol),
the reactant, is much larger compared to the hydration
energy of CH3S- (−65.23 kcal/mol), the product
(the hydration energies of the neutral species are
rather small and have minimal effect on the ∆G of
the reaction). This makes the reaction unfavorable
in the aqueous medium relative to the gas phase
by ~7 kcal/mol.
(B) (IV) S-transfer from CH3SS- and H+ transfer from HCN
It is also possible that the reaction between
CH3SS− to HCN yields CH3SH and SCN−:
CH3SS− + HCN → CH3SH + SCN−. This reaction is
favorable in gas phase (∆E = −27.606 kcal/mol
and ∆G = −27.279 kcal/mol). The formation of a
strong C−S bond in SCN- in place of a weaker
S−S bond in CH3SS− provides the driving force for
this reaction. In water, the reaction is also favorable
(∆E = −24.938 kcal/mol and ∆G = −24.611 kcal/mol).
The calculations presented herein suggest that
biologically important sulfur transfer from thiosulfate
to cyanide is thermodynamically favorable, while
sulfur transfer from thiosulfate to hydrocyanic acid is
unfavorable. The former is due to the formation of a
strong C−S bond in SCN− (product) and the large
solvation energy of SO32− (product) while the latter
is mainly due to the high proton affinity of CN−
(reactant) relative to SCN− (product). This presents
a dilemma because in physiological pH (7.4), HCN
will be present and not CN- as HCN has a pKa of 9.2.
In fact, S−transfer from thiosulfate to CN− could only
be achieved at pH > 10 in vitro.32 Thus, it is evident
that catalysis is required to drive this process under
physiological conditions.
Rhodanese stores the sulfur in a cysteine residue
by converting the catalytic cysteine to cysteine
perthiol. The calculations indicate that this process
also requires the cysteine thiol to be deprotonated.
However, the pKa of thiol is ~10. Thus, in
biologically relevant pHs this residue will be
protonated. In the rhodanese active site, two basic
residues, namely Arg 116 and Arg110, are present in
the active site and they are H-bonded to the catalytic
cysteine to deprotonate the cysteine thiol to make
cysteine thiolate.
The active site resulting after the S-transfer from
thiosulfate will have a perthiolate H-bonded to a
protonated arginine residue (Scheme 1, top). From
here, S-transfer can occur via three pathways: (i) to CN−,
(ii) to HCN, and, (iii) to HCN with a proton
transfer to the resultant thiolate. Thermodynamic
considerations disfavors S-transfer to HCN as it is an
increase of 5.3 kcal/mol. Further, the population of
CN- in pH = 7.4 buffer is < 1 %. This eliminates path
(i). Thus, a synchronous S-transfer from perthiolate
and proton transfer from HCN seems to be the
thermodynamically favored route. However it must
be mentioned that if a 2nd basic site is available to
deprotonate HCN to CN- then the S−transfer can take
place following path (i).
Alternatively, the active site resulting from
S-transfer from thiosulfate may have a perthiol
H-bonded to a neutral arginine residue. Here also,
there are several possible S-transfer routes (Scheme 1,
bottom). However, our calculations indicate that
the PA of CH3SS− is ~8 kcal/mol lower than that
DEY & DEY: DFT CALCULATIONS ON BIOLOGICAL S-TRANSFER REACTIONS
1461
The possible routes for S-transfer from CysSSH to CN- in the 2nd step of the reaction. The favorable steps are indicated by √ while the disfavored
ones are indicated by . ∆Gg and ∆Gs represents free energy for the process in the gas phase and after solvation correction, respectively
×
Scheme 1
Proposed mechanism for S-transfer for perthiol to CN- in the active site of bovine rhodanese
Scheme 2
1462
INDIAN J CHEM, SEC A, SEPT-OCT 2011
of CH3S−. This implies that the pKa of CH3SS− will
be 6 log units lower than that of CH3S−. In fact,
cysteine perthiol is estimated to have a pKa of 6.6 as
against 8.3 in cysteine. Thus, with a lower pKa
than cysteine it is unlikely that cysteine perthiol will
stay protonated in the presence of the same basic
residue that deprotonates cysteine (cysteine has to
be deprotonated to thiolate for S-transfer from
thiosulfate). Thus, the possible S-transfer pathways
involving a cysteine perthiol are likely to be
physiologically irrelevant.
Based on the thermodynamic parameters calculated
for the probable pathways for S-transfer from a
perthiol/perthiolate to HCN/CN− and the relative pKas
of the species involved, the following mechanism
(Scheme 2) can be proposed.33 An active site basic
residue (either of the two conserved arginine residues
H-bonded to the catalytic Cys 254 thiol sulfur of
bovine rhodanese) deprotonates the cysteine thiol to
thiolate, that then accepts an S-atom from thiosulfate
(∆G = −18.9 kcal/mol). This reaction is driven by the
large solvation energy of the resultant SO32− anion.
The cysteine perthiol formed remains deprotonated as
it has a lower pKa. A concerted H+ and S transfer
takes place between HCN and the cysteine
perthiolate. To facilitate this thermodynamically
favored S-atom transfer with synchronous proton
transfer, the active site needs to shuffle protons from
HCN to the resultant thiol. This can be achieved
utilizing the two arginine residues present where one
can abstract the proton from HCN, while the other
simultaneously transfers a proton to the thiolate
formed after S-transfer. Thus, the two acidic arginine
residues that are H-bonded to the cysteine sulfur atom
plays a major role in (a) keeping the active site thiol
deprotonated, and, (b) form a network of H-bonding
which may help proton shuffling in the active site.
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Note that these H-bonding interactions are not modeled in
the current study. These are very important and will be
included in future calculations.