No oxygen isotope exchange between water and APS–sulfate at surface temperature: Evidence from quantum chemical modeling and triple-oxygen isotope experiments

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Geochimica et Cosmochimica Acta 95 (2012) 106–118 www.elsevier.com/locate/gca

No oxygen isotope exchange between water and APS–sulfate at 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 States b 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; available online 2 August 2012

Abstract In both laboratory experiments and natural environments where microbial dissimilatory sulfate reduction (MDSR) occurs in a closed system, the d34 SSO4 ((34S/32S)sample/(34S/32S)standard  1) for dissolved SO42 has been found to follow a typical Rayleigh-Distillation path. In contrast, the corresponding d18 OSO4 ((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 apparent steady-state in the observed difference between d18 OSO4 and d18 OH2 O can be attributed to any of these three steps: (1) the formation of adenosine-50 -phosphosulfate (APS) from ATP and SO42, (2) oxygen exchange between sulfite (or other downstream sulfoxy-anions) and water later in the MDSR reaction chain and its back reaction to APS and sulfate, and (3) the re-oxidation of produced H2S or precursor sulfoxy-anions to sulfate in environments containing Fe(III) or O2. This study examines the first step as a potential pathway for water oxygen incorporation into sulfate. We examined the structures and process of APS formation using B3LYP/6-31G(d,p) hybrid density functional theory, implemented in the Gaussian-03 program suite, to predict the potential for oxygen exchange. We conducted a set of in vitro, enzyme-catalyzed, APS formation experiments (with no further reduction to sulfite) to determine the degree of oxygen isotope exchange between the APS–sulfate and water. Triple-oxygen-isotope labeled water was used in the reactor solutions to monitor oxygen isotope exchange between water and APS sulfate. The formation and hydrolysis of APS were identified as potential steps for oxygen exchange with water to occur. Quantum chemical modeling indicates that the combination of sulfate with ATP has effects on bond strength and symmetry of the sulfate. However, these small effects impart little influence on the integrity of the SO42 tetrahedron due to the high activation energy required for hydrolysis of SO42 (48.94 kcal/mol). Modeling also indicates that APS dissociation via hydrolysis is achieved 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 isotope data of the APS–sulfate indicate no oxygen isotope exchange occurred between APS–sulfate and water at 30 °C for an experimental duration ranging from 2 to 120 h. The study excludes APS formation as one of the causes for sulfate–oxygen isotope exchange with water during MDSR. Ó 2012 Elsevier Ltd. All rights reserved.

1. INTRODUCTION

⇑ Corresponding author.

E-mail address: [email protected] (I.E. Kohl). 0016-7037/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gca.2012.07.018

Microbial dissimilatory sulfate reduction (MDSR) is a ubiquitous process in today’s anoxic Earth surface environments (Widdel, 1988). This process, which transforms

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sulfate to sulfide, derives energy for microbes and is thought to have been an early form of metabolism (Eq. (1)) (Shen and Buick, 2003). 2CH2 O þ SO4 2 ! H2 S þ 2HCO3 

ð1Þ

It has been shown that sulfur and oxygen isotope ratios are powerful parameters for understanding the nature of MDSR. Fritz et al. (1989) showed that the sulfur isotope fractionation during MDSR in a batch experiment follows a Rayleigh-Distillation path: Rsulfate ¼ R0sulfate f a1

ð2Þ

where, Rsulfate = 34S/32S of the remaining sulfate, R0sulfate = 34S/32S of initial sulfate, f = the fraction of sulfate remaining, and a is the isotope fractionation factor and is assumed to remain constant throughout the duration of the experiment process, a ¼ an instantaneous ratio of Rconsumed =Rleft behind

ð3Þ

Thus, the d34 SSO4 of the remaining sulfate increases over time. If sulfate oxygen behaved solely as an integral part of the sulfate tetrahedron, the corresponding d18 OSO4 would increase as well (Turchyn et al., 2010). However, Fritz et al. (1989) found that the d18 OSO4 approaches a steady state value with an apparent enrichment of 23–29& relative to the d18 OH2 O at surface conditions. The behavior of d18 OSO4 suggests that there are certain degrees of oxygen exchange going on between sulfoxyanions and solution water during MDSR. Fritz et al. (1989) proposed that the formation of APS, mediated by the enzyme ATP-sulfurylase (ATPS), from cell internal sulfate and ATP, at the initial stage of MDSR, may weaken the sulfate tetrahedron and result in oxygen isotope exchange with ambient water (Eq. (4); Brunner et al., 2005). They also suggested re-oxidation of intermediate sulfite as a possible exchange pathway (Eq. (5); Brunner et al., 2005; Wortmann et al., 2007; Farquhar et al., 2008; Turchyn et al., 2010). SO4 2 þ ATP

via ATPS APS þ pyrophosphateðPPiÞ

APS

via APS reductase ! SO3

2

þ AMP

ð4Þ ð5Þ

APS generation as a vehicle for oxygen exchange with water has recently reappeared in the literature (Fig. 1). One group of authors used measurements of d18 OSO4 and 18 d OH2 O to test a MDSR model and concluded that enzymecatalyzed, sulfate–water oxygen isotope exchange might indeed be in operation if they could rule out possible sulfite re-oxidation in cytoplasmic water (Brunner and Bernasconi, 2005; Brunner et al., 2005). Farquhar et al. (2008) also see incorporation of water–oxygen in ambient sulfate during sulfate reduction. Their experiments were conducted in an anoxic flow cell reactor and re-oxidation of produced H2S to sulfate is ruled out as a pathway for water–oxygen incorporation into ambient sulfate. Their explanation for this phenomenon is that back reactions between intermediate phases can account for the water–oxygen signal transfer into ambient sulfate. Applying the Brunner and Bernasconi (2005) and Brunner et al. (2005) models to their data, they obtain a best-fit with 78–96% of the ambient sulfate having

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been recycled via backreactions from metabolic sulfoxyanion-intermediates. Based on the sulfur isotope data Farquhar et al. (2008) suggest that SO32 is more likely to be facilitating exchange than APS but note that this is only an inference. A more recent study on batch cultures of sulfate-reducers, in addition to numerical modeling results, suggests that back reactions are indeed favorable but occur to varying degrees depending on the strain of microbes and the reversibility of APS reduction to sulfite (Turchyn et al., 2010). Turchyn et al. (2010) also suggests that, based on both sulfur and oxygen isotope effects, oxygen exchange upstream of APS is unlikely, which serves to further support the need for back reactions to transfer the exchanged sulfoxyanion-intermediates, downstream of APS, back into the ambient sulfate pool. Work done by Wortmann et al. (2007) favors oxygen isotope exchange resulting from an enzymatic reaction (e.g., reverse APS formation from AMP and sulfite), which does not rule out the sulfite exchange/back reaction pathway shown in Fig. 1. Regardless of the pathway of exchange or incorporation of water–oxygen into SO42, all of the above studies postulate some form of steady-state between SO42–oxygen isotopic values and those of ambient water, either through APS formation and decomposition or through a back-reactions to sulfate from intermediate sulfite produced during MDSR, or both. To pin down the exact step that causes sulfate–oxygen isotope exchange with ambient water, we have to confirm or rule out each of the potential steps. Since inorganic sulfite is known to exchange oxygen with water readily (Betts and Voss, 1970; Horner and Connick, 2003), a demonstration that oxygen isotope exchange is not occurring between APS–sulfate and water-will narrow down the exchange steps to a reverse enzymatic reaction from sulfite to sulfate occurring during MDSR. So far, there is little if any theoretical basis for suggesting that APS formation could result in oxygen exchange between APS–sulfate and water. Lalor et al. (2003) suggests that the sulfate tetrahedron is not significantly affected during the enzyme-catalyzed generation of APS. Yet, this continues to appear in the literature (see above) as a potential mechanism for achieving a constant Dd18 OSO4 –H2 O value during MDSR. This study focuses on examining if the enzyme-catalyzed formation of APS from ATP and SO42, the initial step in MDSR process (Fig. 1 and Eq. (4)), results in sulfate–water oxygen isotope exchange. We explore the theoretical basis for oxygen isotope exchange between APS–sulfate and ambient water by modeling structural changes that occur when sulfate is activated to form APS and by determining the location of cleavage that is active during APS dissociation and hydrolysis. Removal of the bridged O-atom (originally incorporated by inorganic sulfate) of the dissociating SO4 group from APS, followed by hydrolysis, could serve as the mechanism of O-exchange with water. We developed model reactions to evaluate such an exchange, which includes both the formation and hydrolysis of APS. Activation energies of corresponding reactions are calculated directly and used as theoretical determinants for the possibility of corresponding exchange channels. Hydrolysis of sulfate and phosphate esters also play a central role in a

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

variety of biochemical processes and modeling such systems is feasible (Rodiguez-Lopez et al., 2001; Akola and Jones, 2003; Wolfenden and Yuan, 2007, and references cited therein). Independently, we examine our model results on the potential for APS–sulfate–water oxygen exchange using in vitro experiments that utilize triple-oxygen-isotope labeled solutions. 2. METHODS 2.1. Modeling APS structure and formation mechanism Theoretical calculations of structural changes in sulfate tetrahedron during APS formation are performed using B3LYP/6-31G(d,p) hybrid density functional theory (DFT) method as implemented in Gaussian-03 suite of programs (Gaussian Inc., Revision D.01, Frisch et al., 2004). The B3LYP method combines the non-local Hartree–Fock exchange functional along with the corrective terms for the density gradient developed by Becke (1993) with the correlation functional by Lee et al. (1988). This hybrid DFTmethod is widely tested in sulfur chemistry and is shown to 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 MP2/6311 + G(3df,2p) wave function method as well as the Onsager solvation model (effect of aqueous media). The interacting system (mediated by the enzyme ATPS reaction of SO42 and ATP4) are prohibitively large for direct potential energy surface (PES) studies. Yet, the general trends can be captured based on computationally more realistic models. Truncated models are particularly effective in the modeling of ATP-related processes (see, e.g., Akola and Jones, 2003; Hansia et al., 2006) and results are generally applicable to a variety of other biologically significant

molecules containing similar linkages. Based on these assumptions, we have developed a straightforward model for the reaction of HSO4 + ATP. It is believed that SO42 is unstable electronically in the isolated state (single ionization is preferred when electronic relaxation is taken into account (Janoschek, 1992; Boldyrev and Simons, 1994; Boldyrev et al., 1996; Zeebe, 2010). Based on this, we chose the interaction of HSO4, which is predominant in low pH (pKa = 1.92), with the ATP possessing different sites of single ionization. To examine the possible effect of pH, we have additionally analyzed the stability of relevant transition states when substrates are fully ionized. The mechanism of the APS formation and hydrolysis reactions are studied using a truncated-phosphosulfate model represented by CH3SP, where the adenosine (adenylyl-ribose, CH2R) part is reduced to CH3-group. Such a model has been successfully used for the modeling of ATP hydrolysis (Akola and Jones, 2003). Transition state structures are optimized using standard TS-search algorithms provided by Gaussian-03 and characterized as having only one negative eigenvalue of the force constant (Hessian) matrices. The absence of imaginary frequencies verifies that structures are true minima. The Intrinsic Reaction Coordinate (IRC) procedure is used for the identification of the connectivity of stationary points on the respective potential energy surfaces (Gonzalez and Schlegel, 1989). The final scan points of IRC are optimized additionally to ensure that reactions from the saddle points lead to the 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-labeled distilled-deionized (DD) water with a D17 OH2 O = 6.00&

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(see below). About 5 mg enzyme-salt NaATPS (Sigma–Aldrich) was added to the ATP + sulfate solution and left to react at 30 °C and neutral pH (pH was 6–7, verified by pH paper prior to addition of ATPS), for 0–120 h to extrapolate a rate of exchange if seen to be occurring. At the conclusion of the experiments, the solution was acidified to pH < 1 with droplets of 10 M HCl to stop the enzyme reaction and to break down the APS. All of the sulfate, ambient and APS, was then precipitated as BaSO4 by addition of saturated BaCl2 to the acidified, degassed solutions. Some BaSO4 was treated twice, using the diethyline-triamine-pentaacetic acid dissolution and re-precipitation (DDARP) technique developed by Bao (2006) to remove ATP that might still be bonded to or occluded in the BaSO4. Experimental duration was varied and experiments were done in duplicate to ensure reproducibility. 2.3. Compound identification by HPLC Verification of APS formation in solution during the course of experiment was achieved through direct high performance liquid chromatography (HPLC) measurements of the acidified and non-acidified samples containing all of the components in question. Analyses were done at LSU Department of Chemistry using an HPLC system equipped with a UV-fluorescence detector. A combination of methanol (4–10 vol.%) and triethylammonium phosphate buffer

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(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 series high performance liquid chromatograph (HPLC) equipped with 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 a diode array detector (Agilent) monitoring ultraviolet absorbance at 254 and 280 nm. Standardization was achieved by running five concentrations of the individual species SO42, ATP, and APS (as NaAPS) and plotting integrated peak area (mAU  s = mill-absorbance units  s) against prepared concentration. All standard calibration curves had r2 values better than 0.98 and standard deviation was between 0.5% and 2% for all runs. In order to test the behavior of the mixtures on peak separation and retention time, mixtures of similar concentrations of SO42, ATP and APS were mixed at 0.1 to 0.0001 M. All sample measurements reported are based on three aliquot measurements of the same sample, each experiment was done in duplicate and therefore generated two samples totaling six replicate measurements. 2.4. Triple-oxygen isotope analysis and the D17O parameter Our experiments utilize triple-oxygen-isotope labeled water. Regardless if oxygen isotope exchange equilibrium

Fig. 2. APS structure calculated at B3LYP/6-31G** hybrid density functional level. Isolated SO42 di-anion structure is inserted for comparison.

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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 as it takes place in APS in regard to the Ribose group (cf. O(3) in Fig. 2). Numbers function as descriptive references.

has been reached, the exact mole fraction of water oxygen in sulfate can be determined from a single experiment via the parameter D17O: D17 OSO4 ¼ mD17 OH2 O 17

ð6Þ 17

was initially calibrated against UWG-2, assuming its d18O = +5.8& (Valley et al., 1995) and its d17O = 0.520  d18O = 3.016&. 3. RESULTS AND DISCUSSION

18

where, the D O  d O  0.52  d O and m is the mole fraction of sulfate oxygen exchanged with the D17O-labeled water. This is valid because the oxygen in the whole system is >99.6% water oxygen (with D17 OH2 O = +6.00&) and SO42 has a very different D17O value (in this case close to zero). This enables m to be defined by one parameter, the D17 OSO4 , as shown above (Eq. (6)), which can be sampled and measured at any given time step during the experiment. Oxygen was generated through a CO2-laser fluorination line on dried BaSO4 (and Na2SO4) powders (Bao and Thiemens, 2000) and was run on a MAT253 isotope-ratio mass spectrometer at LSU. All measurements were done above a certain threshold of gas pressure (20–25 mbar in the bellows) and based on an extrapolation of the VSMOW measurements, assuming ideal linear mass-spectrometric performance (single reference approach). The d17O value

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 affects APS–sulfate symmetry and bond strength and the potential for APS–sulfate to exchange oxygen with water. Structural analysis has been performed for APS2 and SO42 di-anions (Fig. 2) and its truncated form CH3PS2 (Fig. 3) as well as for corresponding mono-anions (not shown due to general similarity) taking into account that at physiological pH they are expected to be completely or mostly ionized. Modeling results show some differences between the symmetrical tetrahedral structure of inorganic sulfate and APS–sulfate. Mulliken population analysis of the

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

symmetric SO42 di-anion indicated on a +1.0 partial charge of the central sulfur atom whereas oxygen atoms share the remaining negative charges at q(O) = 0.75 e. The sulfur atom in APS is more positive (+1.24 e), while negative charge is partly delocalized on the rest of molecule; corresponding O-ligands have less negative charges ranging from 0.63 to 0.68 e. The additional natural bond orbital (NBO)-analysis (Glendening et al., 1998) confirmed this trend. Again, the population of active oxygen atoms decreases in APS as compared with the SO42 di-anion (in average by 0.083 vs. 0.12 e average differences between Mulliken charges). Correspondingly, the positive charge of the central sulfur atom increases in APS (by 0.07 vs. 0.24 e value derived from Mulliken charges indicated above). Terminal oxygen ligands at P-center, in general, are more negative than at the sulfate site (by average 0.11 e of natural charges vs. 0.06 e predicted by Mulliken population analysis), indicating a greater potential to be protonated. As we will see below, such an additional proton affinity causes H-transfer in mono-anion of APS when H2O-molecule is approaching. These structural changes in APS–sulfate relative to inorganic sulfate may well affect the fate of O-ligands of S- and P-tetrahedra in APS–sulfate via the changing of kinetic parameters. We further investigate the formation mechanisms and the effects of hydrolysis via direct calculations for cleavage of S–O and P–O bonds (see below), rather than rely only on the qualitative description of the geometry alteration of the SO42 tetrahedron in APS. 3.1.2. Formation It is believed that APS formation occurs via the pentavalent trigonal bipyramidal transition state (TS), with an inversion of the reaction center: the nucleophilic attack of SO42 on the a-phosphorus center may lead to the inversion of its tetrahedral structure, and removal (cleavage) of

pyrophosphate (see above) (Ullrich et al., 2001). We have modeled this reaction to track the development of O-ligands in sulfate during the formation and decomposition processes. The formation of APS follows the classical Walden inversion mechanism (Lowe, 1991; Alhambra et al., 1998). As expected, such a process is required to overcome a significant activation-energy barrier in the isolated “gasphase” state (220.8 kJ/mol), which is reasonable as it is connected to the inversion of the phosphorous center (for comparison, inversion of the tetrahedral sp3-carbon center faces a ca. 165 kJ/mol activation barrier). However, the reaction becomes facile in biological media due to the catalytic role of the ATP-sulfurylase enzyme, perhaps combined with the supporting solvation effects. Thus, upon formation of APS, the terminal oxygen atom of the sulfate-group forms a bridging double anhydride (S–O–P) bond with phosphategroup (Fig. 4). It is this oxygen atom that has the greatest potential to facilitate oxygen-exchange. Here we note that if the reverse reaction follows the same pathways based on microscopic reversibility of such processes, it would be expected that the dissociated sulfate-group would carry away the same oxygen atom, originally belonging to the inorganic SO42 ion (Fig. 4). 3.1.3. Hydrolysis of APS Here we have developed, to our knowledge, the first direct associative model using high-level quantum chemistry for the hydrolysis of APS mediated by a reactive water molecule approaching either the phosphate or sulfate groups. Corresponding pathways are demonstrated through the relaxed-scan diagrams presented in Figs. 5 and 6. As seen from these energetic profiles, approaching H2O, mediates Walden inversion of reaction centers via the formation of trigonal bipyramidal (TB) transition, similar to the classical SN2-type reactions and the above described formation process of APS.

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Fig. 5. A potential energy surface cross-section for mediated by0 H2O Walden inversion of the PO4-center of the APS0 through a trigonal ˚ and the complete ˚ interatomic O..P distance with the step of 0.1 A bipyramidal transition state. Downward scan started from 3 A optimization of the remaining internal variables resulted in the reaction coordinate R(H2O..P) leading to the formation of a pentacoordinated intermediate. 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 mol1) is higher than the true TS revealed further via the gradient norm optimization of this structure, viz., 37.20 kcal/mol1 (see, Fig. 7) as calculated at the B3LYP/6-31G* hybrid density functional level of theory. DG# = 49.11 kcal mol1 at 298 K. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

TB-intermediates are favored for the reactions of phosphate groups containing substances such as RNA and related compounds. Formation of such intermediates in hydrolysis of methyl ethylene phosphate models, have been studied recently by Uchimaru et al. (1999) using density functional theory. In the case of hydrolysis of the phosphate group of APS, intermolecular interaction also leads to the formation of a pentacoordinated metastable intermediate while hydrolysis of sulfate group leads to direct decomposition to sulfate via TB-transition state. As a result of the hydrolysis of P–O bond, the oxygen atom from the water molecule is retained in the phosphate

part of molecule, as depicted in Fig. 7. The barrier is rather high (ca. 210 kJ/mol; Fig. 5) although it is comparable with the corresponding literature data for “gas-phase” hydrolysis of ATP (146 and 163 kJ/mol depending on mechanism employed; Akola and Jones, 2003). The energy-maximum on this pathway, however, indicates only the approximate position of the reaction barrier, but it is still not the true TS, as one of bonds (reaction coordinate) is fixed. To localize the actual transition state, we have optimized the gradient norm of energy in the vicinity of the maximum point structure, using a procedure implemented in Gaussian-03. Actual TS appears to be localized rather low at

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Fig. 6. A potential energy surface cross-section for mediated by0 H2O Walden inversion of the SO4-center of the APS0 through a trigonal ˚ interatomic O..S distance with the step of 0.1 A ˚ and the complete bipyramidal transition state. Downward scan started from 3 A optimization of the remaining internal variables resulted in the reaction coordinate R(H2O..S) leading to the elimination of the sulfuric acid carrying O-atom of the water molecule (highlighted in red in scheme). The barrier height across this illustrative cross-section (69.1 kcal mol1) is higher than the true TS revealed further via the gradient norm optimization of this structure, viz., 48.94 kcal mol1 (see, Fig. 7), which remains substantially higher than the barrier at PO4-center (see Fig. 5 above). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

DH# = 107.0 kJ/mol (Fig. 7). The intermediate product can undergo further decomposition to sulfate and phosphate. The later molecule will obviously retain the oxygen atom of the water molecule indicating no oxygen-exchange for sulfate (Fig. 5). In contrast, hydrolysis of sulfate initiates direct decomposition to a product set of H2SO4 + CH3PO4H as depicted in Fig. 7. We left a proton on the SO4-group when studying its attack to the a-PO4 tetrahedron of ATP (see below). However, at the start of scan optimization ˚ intermolecular distance) the proton associated with (at 3 A the SO4-group immediately jumps to the proximal oxygen ligand of the P-center. This is not unexpected, as the proton prefers to be added to the O-ligands at P-center from an electrostatic point of view, as described above. The energy of the combined configuration at its maximum point is ca. 80 kJ/mol, higher than in the case of hydrolysis at the P-center.

As indicated above, the maximum point on a scan profile is only a qualitative characteristic of TS, as reaction coordinates (variable intermolecular distance between the oxygen atom of H2O and respective centers P or S) are constrained by definition and the optimized TS will be lower in energy. Indeed, detailed calculations revealed that the true barrier height is 164.7 kJ/mol (Fig. 7). Importantly, the activation barrier for sulfate hydrolysis remains significantly higher (ca. 58 kJ/mol) than its counterpart in the phosphate group, which indicates the domination of phosphate channel, hence confirming the absence of oxygen exchange between sulfate and ambient water. To verify these results we re-calculated two competing TS energies at MP2/6-311 + G(3df.2p) ab initio level. The barrier for PO4-hydrolysis again remains much lower than that for SO4-channel. The difference in electronic energies (zero point vibration energies are close) become even more expressed at 79 kJ/mol.

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

Hydration effects along with the presence of an enzyme, and the acid-alkali catalysis (pH variations) may also alter reaction rates. In this regard, we have performed an analogous analysis for fully ionized substrates (Q = 2), removing only remaining proton at phosphate group from mono-ionized molecules. Corresponding barriers are located at 367.5 and 236.3 kJ/mol for S- and P-tetrahedral inversions, respectively. The activation barriers (relative energies of corresponding maxima) on relaxed scan energy profiles appear to be much higher in doubly ionized systems as compared to mono-ionized species (scan details are not presented here due to the general resemblance). Differences between corresponding hydrolytic mono- and di-ions reactions are 79 and 26 kJ/mol. Importantly, the difference between the two competing channels remains almost the same

at 53 kJ/mol which supports once more the absence of oxygen-exchange between sulfate and water in neutral media. We note that both hydrolysis TS belong to the quasiWalden inversion processes with direct splitting of a water molecule in a trigonal pyramidal TS and surrounding water molecules are expected to participate only as a solvent shell. To ensure that the solvent does not reverse the relative energies of two hydrolysis channels obtained for the gas-phase reactions, we have additionally studied the effect of aqueous media on stabilization of competing transition states using the Onsager solvation model (Wong et al., 1991) based on the self-consistent reaction field (SCRF) method. As expected, the somewhat more polar transition state of SO4-hydrolysis (lTS = 17.1 Debye) appears to be more stabilized than the TS for PO4-channel with the dipole

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

Time (h)

D17O ± 0.05&

APS (mM)

APS stdev

LSUNa2SO4 APS3.3b APS3.4b APS3.5b APS3.6b APS3.7b H2O

0 18 24 76 94 122 0

0.20 0.15 0.23 0.25 0.20 0.24 6.00

N/A 0.729 0.820 1.271 1.222 3.346 N/A

N/A 0.009 0.095 0.024 0.001 0.625 N/A

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

moment of lTS = 14.8 Debye. Nevertheless, the difference amounts only 8.3 kJ/mol, which is insufficient to make changes in energetic preference of phosphate hydrolysis reaction over sulfate channel. The TS for PO4 remains more stable by as much as 49.6 kJ/mol, thus confirming the validity 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 revealed some peak overlaps. However, we were able to determine the APS concentrations (Table 1) based on calibration curves. We determined the production of APS at (2.404 min) on a peak, which was not coincident with any peaks from either SO42 or ATP. The increase of APS concentration (from 0.0007 to 0.003 M) with time was observed (Fig. 8). For ATP, there was difficulty associated with concentration determination in the 1.9–2.1 min region, due to overlap of peaks from all three compounds, APS, ATP, and SO42. The calibration curve generated for the

non-coincident SO42 peak was characterized by small responses in peak area for relatively large changes in concentration [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 standardization was not effective and sulfate concentration data cannot be interpreted from the HPLC results. Thus, [SO42] is not reported here. This made it impossible to use peak area subtraction techniques to back out ATP concentrations. Thus, [ATP] is not reported either. Based on the final APS concentration, 4% of the starting sulfate was APS–sulfate at the time of acidification. 3.2.2. Triple-oxygen-isotope composition of APS–sulfate The triple-oxygen isotope compositions of precipitated BaSO4 and starting Na2SO4 are reported in Table 1. The Na2SO4 and water used in our experiments have a D17O value of 0.20& and 6.00&, respectively. The BaSO4 precipitated from APS–sulfate and ambient sulfate had a D17O between 0.15 and 0.25&, which given the analytical error associated with our triple oxygen isotope measurement

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

technique (±0.05&), indicates that the sulfate precipitated for isotope measurements had the same D17O value as the starting sulfate salt (Fig. 9). The formation of APS in the forward direction via ATPS 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 incubators with pH between 6 and 7 to ensure optimal activity of the enzyme. APS formation in our in vitro experiments is confirmed via HPLC measurements, despite the inability to quantify the other solution components. We acknowledge that the lack of reactant concentration data is not ideal. However, given that the reaction has been shown to proceed under our experimental conditions (Akagi and Campbell, 1962), both forward and backward reactions should have occurred simultaneously in the solution. The D17O values of the experimentally precipitated BaSO4 were statistically invariable for the duration of the experiment (up to 120 h), matching the initial value obtained from the Na2SO4 salt, indicating no oxygen exchange was occurring between sulfate and water. No incorporation of water D17O label into sulfate during in vitro APS synthesis in the presence of the enzyme ATPS indicates that the non-labile nature of sulfate was retained during this reaction. This supports the model results presented in the previous section. ATPS also mediates the APS decomposition reaction (Schmutz and Brunold, 1982). This reverse reaction produces ATP and SO42 from APS and pyrophosphate. Equilibrium kinetics dictates that APS decomposition was occurring during our experiment. This is important for the reintroduction of exchanged SO42 back into the ambient sulfate pool, both in our experiments and in nature. In our case, we are unable to determine the activity of the reverse reaction within our system. However, even if no APS

decomposition occurred prior to acidification, the minimum 4% APS–sulfate in solution would have resulted in +0.06 to +0.24 ± 0.05&, depending on the number of exchanged oxygen molecules (1–4), for the measured D17 OSO4 , which is resolvable analytically but not observed. However, let it be clarified that this 4% is an unrealistically conservative value due to the favorability of the reverse reaction (Schmutz and Brunold, 1982). Additional experiments can look into the kinetics of APS formation and dissociation under a range of solution pH, temperature, or concentration conditions. These kinetic studies can further test the conclusion reached in this work. 4. IMPLICATIONS FOR SULFATE REDUCTION AND RE-OXIDATION In dissimillatory sulfate reducing microbes, ATP is consumed for the production of APS from sulfate. This ATP consumption is compensated by energy gain during degradation of organic matter, which is linked to the reduction of APS to sulfite (electron acceptors), the latter of which is further reduced to hydrogen sulfide. This study indicates that APS formation and dissociation is not a likely step for oxygen isotope exchange between sulfate and water during MDSR. Thus, the reverse process, forming APS from sulfite and AMP, becomes the likely step for causing the apparent oxygen exchange. Note that this reverse step would also require sulfite being oxidized back to sulfate, with 3 oxygen molecules having exchanged with water and the fourth, likely coming from phosphate (Wortmann et al., 2007). The above discussion assumes strictly anoxic conditions, i.e., there is no Fe (III) or O2 in the ambient solution to oxidize sulfite and sulfide that are produced during MDSR. However, many natural environments where MDSR is

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active can contain small amounts of Fe (III) or O2, and a different consortium of microbes (and abiotic pathways) may oxidize sulfite and sulfide back to sulfate. During the re-oxidation, the product sulfate can incorporate oxygen from ambient water via intermediate sulfite. This is also a mechanism by which an oxygen isotope steady-state can be achieved between sulfate in solution and ambient water. At this time, we do not think this possibility can be ruled out as the cause of apparent sulfate–water isotope exchange in many reported cases, especially in natural environments. Our study highlights the need for a better understanding on how forward and backward reactions occur during MDSR and how sulfite or H2S oxidation reactions occur in cell cytoplasm or in environments. 5. CONCLUSIONS Structural modeling of the formation and hydrolysis of APS indicates that some changes occur with respect to the S–O bonds within APS–sulfate structure. However, the non-labile property of the SO42 is retained within APS–sulfate during MDSR. In addition, the dissociation of APS to ATP and SO42 occurs by cleavage of the original P–O bond associated with APS formation, not the S–O bond within sulfate tetrahedron. Thus, there is a lack of mechanistic basis for the ATPS reaction to facilitate sulfatewater oxygen exchange under physiological conditions (neutral pH and ambient temperatures). Meanwhile, triple-oxygen-isotope labeled in vitro experiments confirmed that there is no oxygen isotope exchange between water and APS–sulfate over a 120-h duration. This study precludes APS formation and decomposition as potential steps causing the observed sulfate–water oxygen isotope exchange during MDSR. It suggests, therefore, that two other steps, (1) sulfite–water exchange and back reactions to APS involving AMP or (2) the re-oxidation of produced H2S in solution are responsible. REFERENCES Akagi J. M. and Campbell L. L. (1962) Studies on the thermophilic sulfate-reducing bacteria III. Adenosine triphosphate-sulfurylase of Clostridium nigrificans and Desulfovibrio desulfuricans. J. Bacteriol. 84, 1194–1201. Akola J. and Jones R. O. (2003) Calculation and prediction of molecular structures and reaction paths. SIDE 1, 9. Alhambra C., Wu L, Zhang Z.-Y. and Gao J. (1998) Waldeninversion-enforced transition-state stabilization in a protein tyrosine phosphatase. J. Am. Chem. Soc. 120, 3858. Asatryan R. (2010) Molecular hydrogen assisted transport of Hatoms. Chem. Phys. Lett. 498, 263–269. Asatryan R. and Bozzelli J. W. (2008) Formation of Criegee intermediate in low temperature oxidation of dimethyl sulfoxide. Phys. Chem. 10, 1769–1780. Bao H. (2006) Purifying barite for oxygen isotope measurement by dissolution and reprecipitation in a chelating solution. Anal. Chem. 78, 304–309. Bao H. and Thiemens M. (2000) Generation of O2 from BaSO4 using a CO2-laser fluorination system for the simultaneous analysis of d18O and d17O. Anal. Chem. 72, 4029–4032. Becke A. D. (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648.

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