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Geochimica et Cosmochimica Acta 95 (2012) 106–118

No oxygen isotope exchange between water and APS–sulfateat surface temperature: Evidence from quantum

chemical modeling and triple-oxygen isotope experiments

Issaku E. Kohl a,⇑, Rubik Asatryan b, Huiming Bao a

a Louisiana State University, Department of Geology and Geophysics, E235 Howe-Russell, Geoscience Complex, Baton Rouge, LA 70803,

United Statesb State University of New York, Department of Chemical and Biological Engineering, Buffalo, NY 14226, United States

Received 24 February 2011; accepted in revised form 17 July 2012

Abstract

In both laboratory experiments and natural environments where microbial dissimilatory sulfate reduction (MDSR) occursin a closed system, the d34SSO4

((34S/32S)sample/(34S/32S)standard � 1) for dissolved SO4

2� has been found to follow a typical Ray-leigh-Distillation path. In contrast, the corresponding d18OSO4

((18O/16O)sample/(18O/16O)standard) � 1) is seen to plateau with

an apparent enrichment of between 23& and 29& relative to that of ambient water under surface conditions. This apparentsteady-state in the observed difference between d18OSO4

and d18OH2O can be attributed to any of these three steps: (1) the for-mation of adenosine-50-phosphosulfate (APS) from ATP and SO4

2�, (2) oxygen exchange between sulfite (or other down-stream sulfoxy-anions) and water later in the MDSR reaction chain and its back reaction to APS and sulfate, and (3) there-oxidation of produced H2S or precursor sulfoxy-anions to sulfate in environments containing Fe(III) or O2. This studyexamines the first step as a potential pathway for water oxygen incorporation into sulfate. We examined the structuresand process of APS formation using B3LYP/6-31G(d,p) hybrid density functional theory, implemented in the Gaussian-03program suite, to predict the potential for oxygen exchange. We conducted a set of in vitro, enzyme-catalyzed, APS formationexperiments (with no further reduction to sulfite) to determine the degree of oxygen isotope exchange between the APS–sulfateand water. Triple-oxygen-isotope labeled water was used in the reactor solutions to monitor oxygen isotope exchange betweenwater and APS sulfate.

The formation and hydrolysis of APS were identified as potential steps for oxygen exchange with water to occur. Quantumchemical modeling indicates that the combination of sulfate with ATP has effects on bond strength and symmetry of the sul-fate. However, these small effects impart little influence on the integrity of the SO4

2� tetrahedron due to the high activationenergy required for hydrolysis of SO4

2� (48.94 kcal/mol). Modeling also indicates that APS dissociation via hydrolysis isachieved through cleavage of the P–O bond instead of S–O bond, further supporting the lack of APS–H2O–oxygen exchange.The formation of APS in our in vitro experiments was verified by HPLC fluorescence spectroscopy, and triple-oxygen isotopedata of the APS–sulfate indicate no oxygen isotope exchange occurred between APS–sulfate and water at 30 �C for an exper-imental duration ranging from 2 to 120 h. The study excludes APS formation as one of the causes for sulfate–oxygen isotopeexchange with water during MDSR.� 2012 Elsevier Ltd. All rights reserved.

0016-7037/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.gca.2012.07.018

⇑ Corresponding author.E-mail address: ikohl1@tigers.lsu.edu (I.E. Kohl).

1. INTRODUCTION

Microbial dissimilatory sulfate reduction (MDSR) is aubiquitous process in today’s anoxic Earth surface environ-ments (Widdel, 1988). This process, which transforms

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sulfate to sulfide, derives energy for microbes and isthought to have been an early form of metabolism (Eq.(1)) (Shen and Buick, 2003).

2CH2Oþ SO42� ! H2Sþ 2HCO3

� ð1Þ

It has been shown that sulfur and oxygen isotope ratiosare powerful parameters for understanding the nature ofMDSR. Fritz et al. (1989) showed that the sulfur isotopefractionation during MDSR in a batch experiment followsa Rayleigh-Distillation path:

Rsulfate ¼ R0sulfate f a�1 ð2Þ

where, Rsulfate = 34S/32S of the remaining sulfate,R0

sulfate = 34S/32S of initial sulfate, f = the fraction of sulfateremaining, and a is the isotope fractionation factor and isassumed to remain constant throughout the duration ofthe experiment process,

a ¼ an instantaneous ratio of Rconsumed=Rleft behind ð3Þ

Thus, the d34SSO4of the remaining sulfate increases over

time. If sulfate oxygen behaved solely as an integral partof the sulfate tetrahedron, the corresponding d18OSO4

wouldincrease as well (Turchyn et al., 2010). However, Fritz et al.(1989) found that the d18OSO4

approaches a steady state va-lue with an apparent enrichment of 23–29& relative to thed18OH2O at surface conditions. The behavior of d18OSO4

sug-gests that there are certain degrees of oxygen exchangegoing on between sulfoxyanions and solution water duringMDSR. Fritz et al. (1989) proposed that the formation ofAPS, mediated by the enzyme ATP-sulfurylase (ATPS),from cell internal sulfate and ATP, at the initial stage ofMDSR, may weaken the sulfate tetrahedron and result inoxygen isotope exchange with ambient water (Eq. (4);Brunner et al., 2005). They also suggested re-oxidation ofintermediate sulfite as a possible exchange pathway (Eq.(5); Brunner et al., 2005; Wortmann et al., 2007; Farquharet al., 2008; Turchyn et al., 2010).

SO42� þATP via ATPS

APSþ pyrophosphateðPPiÞ ð4Þ

APS via APS reductase! SO32� þAMP ð5Þ

APS generation as a vehicle for oxygen exchange withwater has recently reappeared in the literature (Fig. 1).

One group of authors used measurements of d18OSO4and

d18OH2O to test a MDSR model and concluded that enzyme-catalyzed, sulfate–water oxygen isotope exchange might in-deed be in operation if they could rule out possible sulfitere-oxidation in cytoplasmic water (Brunner and Bernas-coni, 2005; Brunner et al., 2005). Farquhar et al. (2008) alsosee incorporation of water–oxygen in ambient sulfate dur-ing sulfate reduction. Their experiments were conductedin an anoxic flow cell reactor and re-oxidation of producedH2S to sulfate is ruled out as a pathway for water–oxygenincorporation into ambient sulfate. Their explanation forthis phenomenon is that back reactions between intermedi-ate phases can account for the water–oxygen signal transferinto ambient sulfate. Applying the Brunner and Bernasconi(2005) and Brunner et al. (2005) models to their data, theyobtain a best-fit with 78–96% of the ambient sulfate having

been recycled via backreactions from metabolic sulfoxyan-ion-intermediates. Based on the sulfur isotope data Farqu-har et al. (2008) suggest that SO3

2� is more likely to befacilitating exchange than APS but note that this is onlyan inference. A more recent study on batch cultures of sul-fate-reducers, in addition to numerical modeling results,suggests that back reactions are indeed favorable but occurto varying degrees depending on the strain of microbes andthe reversibility of APS reduction to sulfite (Turchyn et al.,2010). Turchyn et al. (2010) also suggests that, based onboth sulfur and oxygen isotope effects, oxygen exchange up-stream of APS is unlikely, which serves to further supportthe need for back reactions to transfer the exchanged sulf-oxyanion-intermediates, downstream of APS, back intothe ambient sulfate pool.

Work done by Wortmann et al. (2007) favors oxygenisotope exchange resulting from an enzymatic reaction(e.g., reverse APS formation from AMP and sulfite), whichdoes not rule out the sulfite exchange/back reaction path-way shown in Fig. 1. Regardless of the pathway of ex-change or incorporation of water–oxygen into SO4

2, all ofthe above studies postulate some form of steady-state be-tween SO4

2�–oxygen isotopic values and those of ambientwater, either through APS formation and decompositionor through a back-reactions to sulfate from intermediatesulfite produced during MDSR, or both.

To pin down the exact step that causes sulfate–oxygenisotope exchange with ambient water, we have to confirmor rule out each of the potential steps. Since inorganic sul-fite is known to exchange oxygen with water readily (Bettsand Voss, 1970; Horner and Connick, 2003), a demonstra-tion that oxygen isotope exchange is not occurring betweenAPS–sulfate and water-will narrow down the exchangesteps to a reverse enzymatic reaction from sulfite to sulfateoccurring during MDSR. So far, there is little if any theo-retical basis for suggesting that APS formation could resultin oxygen exchange between APS–sulfate and water. Laloret al. (2003) suggests that the sulfate tetrahedron is not sig-nificantly affected during the enzyme-catalyzed generationof APS. Yet, this continues to appear in the literature (seeabove) as a potential mechanism for achieving a constantDd18OSO4–H2O value during MDSR.

This study focuses on examining if the enzyme-catalyzedformation of APS from ATP and SO4

2�, the initial step inMDSR process (Fig. 1 and Eq. (4)), results in sulfate–wateroxygen isotope exchange. We explore the theoretical basisfor oxygen isotope exchange between APS–sulfate andambient water by modeling structural changes that occurwhen sulfate is activated to form APS and by determiningthe location of cleavage that is active during APS dissocia-tion and hydrolysis. Removal of the bridged O-atom (orig-inally incorporated by inorganic sulfate) of the dissociatingSO4 group from APS, followed by hydrolysis, could serveas the mechanism of O-exchange with water. We developedmodel reactions to evaluate such an exchange, which in-cludes both the formation and hydrolysis of APS. Activa-tion energies of corresponding reactions are calculateddirectly and used as theoretical determinants for the possi-bility of corresponding exchange channels. Hydrolysis ofsulfate and phosphate esters also play a central role in a

Fig. 1. Schematic representation of the MDSR reaction chain. Double-ended arrows indicate reversible reactions. Those reactions mediatedby enzymes are shown with the “starred” enzyme mediating the reversible combination of sulfur compounds (squares) with ATP derivedcompounds (triangles). The arrows denoted with “ex” labels represent possible mechanisms for incorporation of water–oxygen throughexchange reactions. (Adapted from Wortmann et al., 2007.)

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variety of biochemical processes and modeling such systemsis feasible (Rodiguez-Lopez et al., 2001; Akola and Jones,2003; Wolfenden and Yuan, 2007, and references citedtherein). Independently, we examine our model results onthe potential for APS–sulfate–water oxygen exchange usingin vitro experiments that utilize triple-oxygen-isotope la-beled solutions.

2. METHODS

2.1. Modeling APS structure and formation mechanism

Theoretical calculations of structural changes in sulfatetetrahedron during APS formation are performed usingB3LYP/6-31G(d,p) hybrid density functional theory(DFT) method as implemented in Gaussian-03 suite of pro-grams (Gaussian Inc., Revision D.01, Frisch et al., 2004).The B3LYP method combines the non-local Hartree–Fockexchange functional along with the corrective terms for thedensity gradient developed by Becke (1993) with the corre-lation functional by Lee et al. (1988). This hybrid DFT-method is widely tested in sulfur chemistry and is shownto be accurate for complex chemical reaction mechanisms(Asatryan and Bozzelli, 2008; Asatryan, 2010). In addition,the relative stability of key transition states of hydrolysis(Section 3.1.3) have been tested using MP2/6-311 + G(3df,2p) wave function method as well as theOnsager solvation model (effect of aqueous media).

The interacting system (mediated by the enzyme ATPSreaction of SO4

2� and ATP4�) are prohibitively large for di-rect potential energy surface (PES) studies. Yet, the generaltrends can be captured based on computationally morerealistic models. Truncated models are particularly effectivein the modeling of ATP-related processes (see, e.g., Akolaand Jones, 2003; Hansia et al., 2006) and results are gener-ally applicable to a variety of other biologically significant

molecules containing similar linkages. Based on theseassumptions, we have developed a straightforward modelfor the reaction of HSO4

� + ATP�.It is believed that SO4

2� is unstable electronically in theisolated state (single ionization is preferred when electronicrelaxation is taken into account (Janoschek, 1992; Boldyrevand Simons, 1994; Boldyrev et al., 1996; Zeebe, 2010).Based on this, we chose the interaction of HSO4

�, whichis predominant in low pH (pKa = 1.92), with the ATP�

possessing different sites of single ionization. To examinethe possible effect of pH, we have additionally analyzedthe stability of relevant transition states when substratesare fully ionized.

The mechanism of the APS formation and hydrolysisreactions are studied using a truncated-phosphosulfatemodel represented by CH3SP, where the adenosine (aden-ylyl-ribose, �CH2R) part is reduced to CH3-group. Sucha model has been successfully used for the modeling ofATP hydrolysis (Akola and Jones, 2003). Transition statestructures are optimized using standard TS-search algo-rithms provided by Gaussian-03 and characterized as hav-ing only one negative eigenvalue of the force constant(Hessian) matrices. The absence of imaginary frequenciesverifies that structures are true minima. The Intrinsic Reac-tion Coordinate (IRC) procedure is used for the identifica-tion of the connectivity of stationary points on therespective potential energy surfaces (Gonzalez and Schlegel,1989). The final scan points of IRC are optimized addition-ally to ensure that reactions from the saddle points lead tothe proper reactants and products.

2.2. APS isotope exchange experiments

About 50 mg of Na2SO4 salt and 50 mg of Na2ATP(both Sigma–Aldrich) were mixed in 5 ml of 17O-labeleddistilled-deionized (DD) water with a D17OH2O = 6.00&

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(see below). About 5 mg enzyme-salt NaATPS (Sigma–Al-drich) was added to the ATP + sulfate solution and left toreact at 30 �C and neutral pH (pH was 6–7, verified by pHpaper prior to addition of ATPS), for 0–120 h to extrapo-late a rate of exchange if seen to be occurring. At the con-clusion of the experiments, the solution was acidified topH < 1 with droplets of 10 M HCl to stop the enzyme reac-tion and to break down the APS. All of the sulfate, ambientand APS, was then precipitated as BaSO4 by addition ofsaturated BaCl2 to the acidified, degassed solutions. SomeBaSO4 was treated twice, using the diethyline-triamine-pen-taacetic acid dissolution and re-precipitation (DDARP)technique developed by Bao (2006) to remove ATP thatmight still be bonded to or occluded in the BaSO4. Experi-mental duration was varied and experiments were done induplicate to ensure reproducibility.

2.3. Compound identification by HPLC

Verification of APS formation in solution during thecourse of experiment was achieved through direct high per-formance liquid chromatography (HPLC) measurements ofthe acidified and non-acidified samples containing all of thecomponents in question. Analyses were done at LSUDepartment of Chemistry using an HPLC system equippedwith a UV-fluorescence detector. A combination of metha-nol (4–10 vol.%) and triethylammonium phosphate buffer

Fig. 2. APS structure calculated at B3LYP/6-31G** hybrid density fucomparison.

(pH �6.0, 90–96 vol.%) was used as the eluent (1 mL/min) in a method modified from Lim and Peters (1989).The specific instrument used was an Agilent 1100 serieshigh performance liquid chromatograph (HPLC) equippedwith a C18 reverse-phase analytical column (Agilent,150 � 4.6 mm) and a C18 guard column (Supelco,50 � 4.6 mm). Analyte detection was achieved using adiode array detector (Agilent) monitoring ultraviolet absor-bance at 254 and 280 nm. Standardization was achieved byrunning five concentrations of the individual species SO4

2�,ATP, and APS (as NaAPS) and plotting integrated peakarea (mAU � s = mill-absorbance units � s) against pre-pared concentration. All standard calibration curves hadr2 values better than 0.98 and standard deviation was be-tween 0.5% and 2% for all runs. In order to test the behav-ior of the mixtures on peak separation and retention time,mixtures of similar concentrations of SO4

2�, ATP andAPS were mixed at 0.1 to 0.0001 M. All sample measure-ments reported are based on three aliquot measurementsof the same sample, each experiment was done in duplicateand therefore generated two samples totaling six replicatemeasurements.

2.4. Triple-oxygen isotope analysis and the D17O parameter

Our experiments utilize triple-oxygen-isotope labeledwater. Regardless if oxygen isotope exchange equilibrium

nctional level. Isolated SO4�2 di-anion structure is inserted for

Fig. 3. Truncated model of APS2� structure, CH3PS2�. The O(13) is also connected via hydrogen bond to a C(3)–H(5) bond of substituent asit takes place in APS in regard to the Ribose group (cf. O(3) in Fig. 2). Numbers function as descriptive references.

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has been reached, the exact mole fraction of water oxygenin sulfate can be determined from a single experiment viathe parameter D17O:

D17OSO4¼ mD17OH2O ð6Þ

where, the D17O � d17O � 0.52 � d18O and m is the molefraction of sulfate oxygen exchanged with the D17O-labeledwater. This is valid because the oxygen in the whole systemis >99.6% water oxygen (with D17OH2O = +6.00&) andSO4

2� has a very different D17O value (in this case closeto zero). This enables m to be defined by one parameter,the D17OSO4

, as shown above (Eq. (6)), which can be sam-pled and measured at any given time step during theexperiment.

Oxygen was generated through a CO2-laser fluorinationline on dried BaSO4 (and Na2SO4) powders (Bao and Thie-mens, 2000) and was run on a MAT253 isotope-ratio massspectrometer at LSU. All measurements were done above acertain threshold of gas pressure (�20–25 mbar in the bel-lows) and based on an extrapolation of the VSMOW mea-surements, assuming ideal linear mass-spectrometricperformance (single reference approach). The d17O value

was initially calibrated against UWG-2, assuming itsd18O = +5.8& (Valley et al., 1995) and itsd17O = 0.520 � d18O = 3.016&.

3. RESULTS AND DISCUSSION

3.1. Theoretical modeling

3.1.1. APS structure

Structural models of SO42� and APS2� were studied

(Figs. 2 and 3) in order to determine how APS synthesis af-fects APS–sulfate symmetry and bond strength and the po-tential for APS–sulfate to exchange oxygen with water.Structural analysis has been performed for APS2� andSO4

2� di-anions (Fig. 2) and its truncated form CH3PS2�

(Fig. 3) as well as for corresponding mono-anions (notshown due to general similarity) taking into account thatat physiological pH they are expected to be completely ormostly ionized.

Modeling results show some differences between thesymmetrical tetrahedral structure of inorganic sulfate andAPS–sulfate. Mulliken population analysis of the

Fig. 4. A simplified model for APS formation via the trigonal bipyramidal TS structure calculated at B3LYP/6-31G** hybrid densityfunctional level.

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symmetric SO42� di-anion indicated on a +1.0 partial

charge of the central sulfur atom whereas oxygen atomsshare the remaining negative charges at q(O) = �0.75 e.The sulfur atom in APS is more positive (+1.24 e), whilenegative charge is partly delocalized on the rest of molecule;corresponding O-ligands have less negative charges rangingfrom �0.63 to �0.68 e. The additional natural bond orbital(NBO)-analysis (Glendening et al., 1998) confirmed thistrend. Again, the population of active oxygen atoms de-creases in APS as compared with the SO4

2� di-anion (inaverage by 0.083 vs. 0.12 e average differences betweenMulliken charges). Correspondingly, the positive chargeof the central sulfur atom increases in APS (by 0.07 vs.0.24 e value derived from Mulliken charges indicatedabove).

Terminal oxygen ligands at P-center, in general, aremore negative than at the sulfate site (by average 0.11 eof natural charges vs. 0.06 e predicted by Mulliken popula-tion analysis), indicating a greater potential to be proton-ated. As we will see below, such an additional protonaffinity causes H-transfer in mono-anion of APS whenH2O-molecule is approaching.

These structural changes in APS–sulfate relative to inor-ganic sulfate may well affect the fate of O-ligands of S- andP-tetrahedra in APS–sulfate via the changing of kineticparameters. We further investigate the formation mecha-nisms and the effects of hydrolysis via direct calculationsfor cleavage of S–O and P–O bonds (see below), rather thanrely only on the qualitative description of the geometryalteration of the SO4

2� tetrahedron in APS.

3.1.2. Formation

It is believed that APS formation occurs via the pentava-lent trigonal bipyramidal transition state (TS), with aninversion of the reaction center: the nucleophilic attack ofSO4

2� on the a-phosphorus center may lead to the inver-sion of its tetrahedral structure, and removal (cleavage) of

pyrophosphate (see above) (Ullrich et al., 2001). We havemodeled this reaction to track the development of O-li-gands in sulfate during the formation and decompositionprocesses.

The formation of APS follows the classical Waldeninversion mechanism (Lowe, 1991; Alhambra et al., 1998).As expected, such a process is required to overcome a sig-nificant activation-energy barrier in the isolated “gas-phase” state (220.8 kJ/mol), which is reasonable as it is con-nected to the inversion of the phosphorous center (for com-parison, inversion of the tetrahedral sp3-carbon center facesa ca. 165 kJ/mol activation barrier). However, the reactionbecomes facile in biological media due to the catalytic roleof the ATP-sulfurylase enzyme, perhaps combined with thesupporting solvation effects. Thus, upon formation of APS,the terminal oxygen atom of the sulfate-group forms abridging double anhydride (S–O–P) bond with phosphate-group (Fig. 4). It is this oxygen atom that has the greatestpotential to facilitate oxygen-exchange. Here we note thatif the reverse reaction follows the same pathways basedon microscopic reversibility of such processes, it would beexpected that the dissociated sulfate-group would carryaway the same oxygen atom, originally belonging to theinorganic SO4

2� ion (Fig. 4).

3.1.3. Hydrolysis of APS

Here we have developed, to our knowledge, the first di-rect associative model using high-level quantum chemistryfor the hydrolysis of APS mediated by a reactive water mol-ecule approaching either the phosphate or sulfate groups.Corresponding pathways are demonstrated through the re-laxed-scan diagrams presented in Figs. 5 and 6. As seenfrom these energetic profiles, approaching H2O, mediatesWalden inversion of reaction centers via the formation oftrigonal bipyramidal (TB) transition, similar to the classicalSN2-type reactions and the above described formation pro-cess of APS.

Fig. 5. A potential energy surface cross-section for mediated by H2O Walden inversion of the PO4-center of the APS through a trigonalbipyramidal transition state. Downward scan started from 3 A

0interatomic O..P distance with the step of �0.1 A

0and the complete

optimization of the remaining internal variables resulted in the reaction coordinate R(H2O..P) leading to the formation of a pentacoordinatedintermediate. As seen, an oxygen atom of the water molecule (highlighted in red in the bottom scheme) is embedded in the phosphate residue.The barrier height across this illustrative cross-section (50.2 kcal mol�1) is higher than the true TS revealed further via the gradient normoptimization of this structure, viz., 37.20 kcal/mol�1 (see, Fig. 7) as calculated at the B3LYP/6-31G* hybrid density functional level of theory.DG# = 49.11 kcal mol�1 at 298 K. (For interpretation of the references to colour in this figure legend, the reader is referred to the web versionof this article.)

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TB-intermediates are favored for the reactions of phos-phate groups containing substances such as RNA and re-lated compounds. Formation of such intermediates inhydrolysis of methyl ethylene phosphate models, have beenstudied recently by Uchimaru et al. (1999) using densityfunctional theory. In the case of hydrolysis of the phos-phate group of APS, intermolecular interaction also leadsto the formation of a pentacoordinated metastable interme-diate while hydrolysis of sulfate group leads to directdecomposition to sulfate via TB-transition state.

As a result of the hydrolysis of P–O bond, the oxygenatom from the water molecule is retained in the phosphate

part of molecule, as depicted in Fig. 7. The barrier is ratherhigh (ca. 210 kJ/mol; Fig. 5) although it is comparable withthe corresponding literature data for “gas-phase” hydroly-sis of ATP (146 and 163 kJ/mol depending on mechanismemployed; Akola and Jones, 2003). The energy-maximumon this pathway, however, indicates only the approximateposition of the reaction barrier, but it is still not the trueTS, as one of bonds (reaction coordinate) is fixed. To local-ize the actual transition state, we have optimized the gradi-ent norm of energy in the vicinity of the maximum pointstructure, using a procedure implemented in Gaussian-03.Actual TS appears to be localized rather low at

Fig. 6. A potential energy surface cross-section for mediated by H2O Walden inversion of the SO4-center of the APS through a trigonalbipyramidal transition state. Downward scan started from 3 A

0interatomic O..S distance with the step of �0.1 A

0and the complete

optimization of the remaining internal variables resulted in the reaction coordinate R(H2O..S) leading to the elimination of the sulfuric acidcarrying O-atom of the water molecule (highlighted in red in scheme). The barrier height across this illustrative cross-section (69.1 kcal mol�1)is higher than the true TS revealed further via the gradient norm optimization of this structure, viz., 48.94 kcal mol�1 (see, Fig. 7), whichremains substantially higher than the barrier at PO4-center (see Fig. 5 above). (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

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DH# = 107.0 kJ/mol (Fig. 7). The intermediate product canundergo further decomposition to sulfate and phosphate.The later molecule will obviously retain the oxygen atomof the water molecule indicating no oxygen-exchange forsulfate (Fig. 5).

In contrast, hydrolysis of sulfate initiates direct decom-position to a product set of H2SO4 + CH3PO4H� as de-picted in Fig. 7. We left a proton on the SO4

�-groupwhen studying its attack to the a-PO4 tetrahedron ofATP (see below). However, at the start of scan optimization(at 3 A intermolecular distance) the proton associated withthe SO4

�-group immediately jumps to the proximal oxygenligand of the P-center. This is not unexpected, as the protonprefers to be added to the O-ligands at P-center from anelectrostatic point of view, as described above. The energyof the combined configuration at its maximum point isca. 80 kJ/mol, higher than in the case of hydrolysis at theP-center.

As indicated above, the maximum point on a scan pro-file is only a qualitative characteristic of TS, as reactioncoordinates (variable intermolecular distance between theoxygen atom of H2O and respective centers P or S) are con-strained by definition and the optimized TS will be lower inenergy. Indeed, detailed calculations revealed that the truebarrier height is 164.7 kJ/mol (Fig. 7). Importantly, theactivation barrier for sulfate hydrolysis remains signifi-cantly higher (ca. 58 kJ/mol) than its counterpart in thephosphate group, which indicates the domination of phos-phate channel, hence confirming the absence of oxygen ex-change between sulfate and ambient water.

To verify these results we re-calculated two competingTS energies at MP2/6-311 + G(3df.2p) ab initio level. Thebarrier for PO4-hydrolysis again remains much lower thanthat for SO4-channel. The difference in electronic energies(zero point vibration energies are close) become even moreexpressed at 79 kJ/mol.

Fig. 7. Actual transition state (TS) structures for two competitive hydrolysis pathways, viz., through attack of water molecule to thephosphate and sulfate reaction centers, respectively. Due to the substantial difference in activation barriers, hydrolysis at sulfate centerresulting in the oxygen–atom exchange with ambient water is predicted to be much less favored.

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Hydration effects along with the presence of an enzyme,and the acid-alkali catalysis (pH variations) may also alterreaction rates. In this regard, we have performed ananalogous analysis for fully ionized substrates (Q = �2),removing only remaining proton at phosphate group frommono-ionized molecules. Corresponding barriers are lo-cated at 367.5 and 236.3 kJ/mol for S- and P-tetrahedralinversions, respectively. The activation barriers (relativeenergies of corresponding maxima) on relaxed scan energyprofiles appear to be much higher in doubly ionized systemsas compared to mono-ionized species (scan details are notpresented here due to the general resemblance). Differencesbetween corresponding hydrolytic mono- and di-ions reac-tions are 79 and 26 kJ/mol. Importantly, the difference be-tween the two competing channels remains almost the same

at 53 kJ/mol which supports once more the absence of oxy-gen-exchange between sulfate and water in neutral media.

We note that both hydrolysis TS belong to the quasi-Walden inversion processes with direct splitting of a watermolecule in a trigonal pyramidal TS and surrounding watermolecules are expected to participate only as a solvent shell.To ensure that the solvent does not reverse the relative ener-gies of two hydrolysis channels obtained for the gas-phasereactions, we have additionally studied the effect of aqueousmedia on stabilization of competing transition states usingthe Onsager solvation model (Wong et al., 1991) based onthe self-consistent reaction field (SCRF) method.

As expected, the somewhat more polar transition stateof SO4-hydrolysis (lTS = 17.1 Debye) appears to be morestabilized than the TS for PO4-channel with the dipole

Table 1Experimental data (averages) from three aliquot measurements of the same sample; each experiment was done in duplicate (six totalmeasurements) at pH 7 and 30 �C.

Sample Time (h) D17O ± 0.05& APS (mM) APS stdev

LSUNa2SO4 0 �0.20 N/A N/AAPS3.3b 18 �0.15 0.729 0.009APS3.4b 24 �0.23 0.820 0.095APS3.5b 76 �0.25 1.271 0.024APS3.6b 94 �0.20 1.222 0.001APS3.7b 122 �0.24 3.346 0.625H2O 0 6.00 N/A N/A

Fig. 8. APS concentrations changing with time based on three aliquot measurements of the same sample; each experiment was done induplicate. Standard deviations are reported in Table 1. Some error bars are smaller than the size of symbols.

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moment of lTS = 14.8 Debye. Nevertheless, the differenceamounts only 8.3 kJ/mol, which is insufficient to makechanges in energetic preference of phosphate hydrolysisreaction over sulfate channel. The TS for PO4 remains morestable by as much as 49.6 kJ/mol, thus confirming the valid-ity of above conclusions based on gas-phase models.

3.2. In vitro experiments

3.2.1. Compound identification by HPLC

HPLC examination of the experimental solutions re-vealed some peak overlaps. However, we were able to deter-mine the APS concentrations (Table 1) based on calibrationcurves. We determined the production of APS at(2.404 min) on a peak, which was not coincident with anypeaks from either SO4

2� or ATP. The increase of APS con-centration (from 0.0007 to 0.003 M) with time was observed(Fig. 8). For ATP, there was difficulty associated with con-centration determination in the 1.9–2.1 min region, due tooverlap of peaks from all three compounds, APS, ATP,and SO4

2�. The calibration curve generated for the

non-coincident SO42� peak was characterized by small re-

sponses in peak area for relatively large changes in concen-tration [y (peak area) = 0.0003x (concentration) � 0.0529].This observation, coupled with the non-zero intercept(y = �0.0529 for x = 0), indicates that sulfate standardiza-tion was not effective and sulfate concentration data cannotbe interpreted from the HPLC results. Thus, [SO4

2�] is notreported here. This made it impossible to use peak area sub-traction techniques to back out ATP concentrations. Thus,[ATP] is not reported either. Based on the final APS con-centration, 4% of the starting sulfate was APS–sulfate atthe time of acidification.

3.2.2. Triple-oxygen-isotope composition of APS–sulfate

The triple-oxygen isotope compositions of precipitatedBaSO4 and starting Na2SO4 are reported in Table 1. TheNa2SO4 and water used in our experiments have a D17O va-lue of �0.20& and 6.00&, respectively. The BaSO4 precip-itated from APS–sulfate and ambient sulfate had a D17Obetween �0.15 and �0.25&, which given the analytical er-ror associated with our triple oxygen isotope measurement

Fig. 9. The D17O label contained in the experimental waters (red squares) and the D17O of BaSO4 precipitated from aqueous sulfate (bluediamonds). Also plotted are the analyses of Sigma–Aldrich Na2SO4 (orange triangles). (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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technique (±0.05&), indicates that the sulfate precipitatedfor isotope measurements had the same D17O value as thestarting sulfate salt (Fig. 9).

The formation of APS in the forward direction viaATPS has been known to occur in the pH range 6.0–9.5,with activity ceasing below pH 5 (Akagi and Campbell,1962). Our experiments were conducted in 30� C incubatorswith pH between 6 and 7 to ensure optimal activity of theenzyme. APS formation in our in vitro experiments is con-firmed via HPLC measurements, despite the inability toquantify the other solution components. We acknowledgethat the lack of reactant concentration data is not ideal.However, given that the reaction has been shown to pro-ceed under our experimental conditions (Akagi and Camp-bell, 1962), both forward and backward reactions shouldhave occurred simultaneously in the solution.

The D17O values of the experimentally precipitatedBaSO4 were statistically invariable for the duration of theexperiment (up to 120 h), matching the initial value ob-tained from the Na2SO4 salt, indicating no oxygen ex-change was occurring between sulfate and water. Noincorporation of water D17O label into sulfate duringin vitro APS synthesis in the presence of the enzyme ATPSindicates that the non-labile nature of sulfate was retainedduring this reaction. This supports the model results pre-sented in the previous section.

ATPS also mediates the APS decomposition reaction(Schmutz and Brunold, 1982). This reverse reaction pro-duces ATP and SO4

2� from APS and pyrophosphate. Equi-librium kinetics dictates that APS decomposition wasoccurring during our experiment. This is important forthe reintroduction of exchanged SO4

2� back into the ambi-ent sulfate pool, both in our experiments and in nature. Inour case, we are unable to determine the activity of the re-verse reaction within our system. However, even if no APS

decomposition occurred prior to acidification, the mini-mum 4% APS–sulfate in solution would have resulted in+0.06 to +0.24 ± 0.05&, depending on the number of ex-changed oxygen molecules (1–4), for the measuredD17OSO4

, which is resolvable analytically but not observed.However, let it be clarified that this 4% is an unrealisticallyconservative value due to the favorability of the reversereaction (Schmutz and Brunold, 1982). Additional experi-ments can look into the kinetics of APS formation and dis-sociation under a range of solution pH, temperature, orconcentration conditions. These kinetic studies can furthertest the conclusion reached in this work.

4. IMPLICATIONS FOR SULFATE REDUCTION AND

RE-OXIDATION

In dissimillatory sulfate reducing microbes, ATP is con-sumed for the production of APS from sulfate. This ATPconsumption is compensated by energy gain during degra-dation of organic matter, which is linked to the reductionof APS to sulfite (electron acceptors), the latter of whichis further reduced to hydrogen sulfide. This study indicatesthat APS formation and dissociation is not a likely step foroxygen isotope exchange between sulfate and water duringMDSR. Thus, the reverse process, forming APS from sul-fite and AMP, becomes the likely step for causing theapparent oxygen exchange. Note that this reverse stepwould also require sulfite being oxidized back to sulfate,with 3 oxygen molecules having exchanged with waterand the fourth, likely coming from phosphate (Wortmannet al., 2007).

The above discussion assumes strictly anoxic conditions,i.e., there is no Fe (III) or O2 in the ambient solution to oxi-dize sulfite and sulfide that are produced during MDSR.However, many natural environments where MDSR is

I.E. Kohl et al. / Geochimica et Cosmochimica Acta 95 (2012) 106–118 117

active can contain small amounts of Fe (III) or O2, and adifferent consortium of microbes (and abiotic pathways)may oxidize sulfite and sulfide back to sulfate. During there-oxidation, the product sulfate can incorporate oxygenfrom ambient water via intermediate sulfite. This is also amechanism by which an oxygen isotope steady-state canbe achieved between sulfate in solution and ambient water.At this time, we do not think this possibility can be ruledout as the cause of apparent sulfate–water isotope exchangein many reported cases, especially in natural environments.Our study highlights the need for a better understanding onhow forward and backward reactions occur during MDSRand how sulfite or H2S oxidation reactions occur in cellcytoplasm or in environments.

5. CONCLUSIONS

Structural modeling of the formation and hydrolysis ofAPS indicates that some changes occur with respect tothe S–O bonds within APS–sulfate structure. However,the non-labile property of the SO4

2� is retained withinAPS–sulfate during MDSR. In addition, the dissociationof APS to ATP and SO4

2� occurs by cleavage of the origi-nal P–O bond associated with APS formation, not the S–Obond within sulfate tetrahedron. Thus, there is a lack ofmechanistic basis for the ATPS reaction to facilitate sul-fate�water oxygen exchange under physiological condi-tions (neutral pH and ambient temperatures). Meanwhile,triple-oxygen-isotope labeled in vitro experiments confirmedthat there is no oxygen isotope exchange between water andAPS–sulfate over a 120-h duration. This study precludesAPS formation and decomposition as potential steps caus-ing the observed sulfate–water oxygen isotope exchangeduring MDSR. It suggests, therefore, that two other steps,(1) sulfite–water exchange and back reactions to APSinvolving AMP or (2) the re-oxidation of produced H2Sin solution are responsible.

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Associate editor: James Farquhar