Calculation of the aqueous thermodynamic properties of citric acid cycle intermediates and precursors and the estimation of high temperature and pressure equation of state parameters.

The citric acid cycle (CAC) is the central pathway of energy transfer for many organisms, and understanding the origin of this pathway may provide insight into the origins of metabolism. In order to assess the thermodynamics of this key pathway for microorganisms that inhabit a wide variety of environments, especially those found in high temperature environments, we have calculated the properties and parameters for the revised Helgeson-Kirkham-Flowers equation of state for the major components of the CAC. While a significant amount of data is not available for many of the constituents of this fundamental pathway, methods exist that allow estimation of these missing data.

Introduction

The citric acid cycle (CAC), also known as the Krebs or tricarboxylic acid cycle, is a fundamental pathway in intermediary metabolism among all the domains of life. Organisms that use the CAC (or especially the reverse or reductive CAC) are represented among the most deeply-rooted autotrophic hyperthermophilic Archaea and Bacteria and the most derived of organotrophic evolutionary lineages [1-4]. The key role of iron-sulfur proteins, thioester intermediates, and the reductive use of the CAC in hyperthermophilic autotrophs link the CAC to prebiotic theories of energy metabolism and abiotic carbon fixation at deep-sea hydrothermal vents [5-7]. Because of the fundamental metabolic roles and evolutionary importance of the CAC and the reverse CAC, it is of interest to understand the conditions constraining the many reactions of which it may be composed under the considerable range of physical and chemical environments where it functions. Most thermodynamic data available for the substrates used in the CAC are incomplete and have only been determined at the 25 °C and 0.1 MPa standard state. Therefore it is difficult to predict accurate reaction thermodynamics well beyond those conditions, including those likely to have hosted the emergence of life [7-10]. For examination of CAC reactions under the relatively extreme high-temperature and high-pressure conditions where life can thrive and may have originated, and to determine the geochemical environments where prebiotic conditions may have been favorable, the high pressure and temperature thermodynamic parameters of the substrates must be determined.

To evaluate the standard Gibbs free energy ( ) of reaction for a given set of products and reactants at temperatures and pressures beyond the 25 °C – 0.1 MPa reference conditions in an aqueous system, the standard Gibbs free energy of formation ( ) for each substance at non-standard temperature and pressure must be calculated. Most thermodynamic data available for aqueous organic molecules beyond the 25 °C and 0.1 MPa reference conditions are generally for compounds that are either potential growth substrates or metabolic by-products. Only in a few works are there data available for compounds that are intermediates in the ubiquitous biochemical pathways found in nature. The general lack of empirical data for aqueous organic compounds at elevated temperature and pressure has lead to the development of various equations of state to describe the behavior of various thermodynamic properties at non-standard temperature and pressure [11,12]. The revised Helgeson-Kirkham-Flowers equations of state [11,13], along with methods for the estimation of high pressure and temperature thermodynamic properties [14-20], can be used to predict accurately reaction thermochemical properties for aqueous organic compounds well within the thermal and pressure range of the possible environments where these reactions may be expected to occur in biological processes. In addition to aqueous organic compounds [13,18,21,22], the revised HKF equations of state have been used to calculate accurately the thermodynamic reaction properties, without the benefit of high pressure and temperature data, for aqueous inorganic [17] and organic ions [13,23], and inorganic electrolytes [19] at subcritical temperatures and pressures up to 500 MPa. Such versatility makes it one of the more useful tools for evaluating geochemical and biochemical reaction properties in a wide variety of environments, as the revised HKF allows calculation of species and reaction properties among minerals, gases and aqueous species. This provides a framework for understanding many interdependent metabolic and geochemical processes [24].

Data for reactions depicted in Figure 1 are used as a suggestion for a prebiotic reductive CAC. However, estimation of the thermochemical properties of the thioester intermediates may also function as proxies in lieu of high pressure and temperature reaction data for coenzyme A or other thioester intermediates found in Archaea.

Figure 1.

The reverse citric acid cycle (modified from [25]).

Methods

The revised HKF equation of state uses the standard [26] , aqueous partial molal entropy (S0aq), partial molal volume (V0), and constant pressure molal heat capacity ( ), along with fitting parameters that integrate the change in the partial molal property, into the at the desired temperature and pressure conditions. In regard to the revised- HKF equation of state, the 25 °C – 0.1 MPa properties are referred to as the reference state, i.e. , and the calculated partial molal property at P and T, the standard state, i.e. . For substances for which incomplete thermodynamic data are available, the use of group contribution, or additivity, algorithms has become the most pragmatic method available in light of the vast number of naturally occurring and synthetic organic compounds. Group additivity relationships have been used to generate 25 °C, 0.1 MPa reference state thermodynamic properties and group values. These estimation methods have been used for pure phase gas, liquid and solid organic compounds [27-29] and aqueous neutral and ionic organic compounds. Namely among the aqueous species are n-alkanes, n-alkenes, n-alcohols, n–alkanones [30], aldehydes [15], amino acids [18,22], carboxylic, hydroxy and dicarboxylic acids, and their respective ions [23], as well as numerous biochemically-relevant organic compounds (e.g.[31,32]).

Strategy used for the estimation of missing reference state values

The approach taken to estimate reference state values herein is to utilize methods using state variables and reaction data, where available, to calculate missing values. In absence of state or reaction data, the group contribution method is used, with emphasis on attaining data for the closest structural analogues as a base structure. In doing so, the fewest group values are used to modify the base structure as a means of decreasing the probability of error accumulation. The selection of methods used to estimate reference state data are summarized in Figure 2 using the neutral species as examples.

Figure 2.

Group contribution method used to estimate (a) the standard partial molal entropy (S°) and volume (V°) and (b) the heat capacity (Cp°) and free energy (ΔGf°) of aqueous organic species considered in this study.

a-value taken from reference shown in Table 1. b- group values from [35]. c- the carbonyl group value was estimated from the difference in V0 between succinic acid [23] and α-ketoglutarate [36]. d-estimated using group values from [31]. e-malonic acid from [23]. f-group value for >C=O assumed to be the difference in between α-ketoglutaric acid [37] and succinic acid [23]. g-calculated form pKa values from [33] and for ions from Table 1 (as described in text). h-value for was taken from [38], the was from [33], and were the CODATA values from [39]. i-estimated from V0 of H-fumarate−1 (Figure 2) assuming the same difference in V0 between the acid and ion as between succinic acid and H-succinate−1 from [23]. j-estimated by subtracting the difference in y-intercept values for between n-alkanes and n-alkenes (taken from [18]) from that of succinic acid (taken from [23]). k- calculated from pKa and values from [33] for ions (as described in text), was from [37)] and were the CODATA values from [39]. l-the for succinic acid was taken from [23]. The group value for the >C=O was taken from [31]. m-from [18]. n-group value estimated from difference in values between ethyl sulfide (as described in Appendix) and n-butane (taken from [18]). o- group value from [40]. p- group value estimated from difference in values between ethyl sulfide [34] and n-butane (taken from [18]. q-from [23]. r-group value estimated from difference in y-intercepts of between n-alkanones and n-1 n-alkanes taken from [18]. s- group value from [30]. t- group value estimated from difference in y-intercepts of between n-alkanones and n-1 n-alkanes taken from [18]. u- group value estimated from difference in y-intercepts of between n-alkanones and n-1 n-alkanes taken from [18]. v- group value estimated from difference in values between ethyl sulfide (calculated from and Δ from [34] and n-butane (taken from [18)].

Calculation of reference state and estimation of missing values

The values selected for neutral and ionic organic species are shown in Table 1. The for organic acids and ions for which there were no data available were calculated from the ionization constants (pKa), critically compiled in [33], and of the respective ion or acid for which there were reliable data. The only acid-anion pair for which values were unavailable was succinyl thioester. Therefore, estimation of its was necessary and attained through the addition of the for the (>C=O) and (–S-) groups to pentanoic acid-pentanoate for the succinyl thioester acid-anion pair. The group value was calculated from the difference in the y-intercepts for regression equations between n-alkanones and (n-1) n-alkanes (both taken from reference [18]). The was calculated similarly, from the difference in y-intercepts from dialkylsulfides [34] and n-alkanes [18], with the same number of carbon atoms. Data for other components of the citric acid cycle (ethyl thiol and acetic acid; see Figure 1) are available in the literature [16,23].

Table 1.

Summary of the aqueous reference state (25 °C, 0.1 MPa) partial molal properties of organic species and parameters for the revised HKF equations of state used to extrapolate to elevated temperatures and pressures.

	ΔGf0a	S0b	V0c	CP0b	a1d,ee	a2d	a3e	a4f,hh	c1g	c2f	ωb
pyruvic acid	−489.1j	179.9 k	54.6 h	114.6 h	3.9096	5745.0 ff	2.478	−140079	24.645	384086 jj	−129222 mm
pyruvate−1	−474.9 I	171.5 k	41.5 t	−52.8 bb	3.2601	4220.3 ff	17.617	−133776	22.278	−178281 ii	42208 ll
oxalaoacetic acid	−838.3 j	287.5 h	72.4 h	108.7 h	4.8998	7178.9 ff	24.687	−146006	27.207	377592 jj	−52158 mm
H-oxalaoacetate−1	−823.7 j	233.1 p	60.3 u	−77.7 cc	4.3045	6672.1 ff	10.473	−143911	−10.600	−181300 ii	330604 ll
oxalaoacetate−2	−798.7 I	107.9 q	46.7 v	−328.1 cc	3.8109	5513.3 ff	13.850	−139121	−176.230	−211639 ii	1139579 ll
malic acid	−891.6 k	283.8 h	82.8 w	227.7 w	5.5023	8481.8 ff	21.621	−151393	117.243	509492 jj	−75348 mm
H-malate−1	−872.4 l	227.7 r	69.4 x	41.2 dd	4.8234	7890.2 ff	6.924	−148947	106.200	−166880 ii	344473 ll
malate−2	−843.1 l	126.8 r	55.7 x	−209.1 dd	4.3174	6702.4 ff	10.385	−144037	−62.817	−197220 ii	1170226 ll
fumaric acid	−645.8 k	261.1 k	78.8 h	154.7 h	5.2807	8963.5 ff	39.867	−153384	58.786	428516 jj	−95650 mm
H-fumarate−1	−628.1 k	203.3 k	65.4 y	−31.8 dd	4.6074	7383.1 ff	8.401	−146851	38.350	−175733 ii	373932 ll
fumarate−2	−601.9 k	105.4 k	51.7 y	−282.1 dd	4.0998	6191.7 ff	11.873	−141925	−131.082	−206072 ii	1185066 ll
α-ketoglutaric acid	−842.3 j	315.1 h	89.0 z	173.4 h	5.8421	9216.6 ff	24.128	−154430	78.884	449296 jj	−35121 mm
H-α-ketoglutarate−1	−829.4 j	243.5 s	75.6 x	−13.1 dd	5.1687	8700.7 ff	4.562	−152298	51.025	−173461 ii	292131 ll
α-ketoglutarate−2	−802.0 I	136.0 s	61.9 x	−263.4 dd	4.6661	7520.8 ff	8.000	−147420	−117.057	−203800 ii	1117934 ll
citric acid	−1243.4 m	329.4 l	113.6 w	322.5 w	7.2438	12247.7 ff	39.901	−166961	195.456	614557 jj	−23333 mm
H2-citrate−1	−1226.3 k	286.2 k	98.1 aa	187.9 k	6.4344	11671.9 ff	−4.096	−164580	241.056	−149109 ii	248464 ll
H-citrate−2	−1199.2 k	202.3 k	88.5 aa	0.84 k	6.1522	11009.3 ff	−2.165	−161841	131.407	−171775 ii	1038339 ll
citrate−3	−1162.7 k	92.1 k	72.0 aa	−254.8 k	5.4914	9458.3 ff	2.355	−155429	−40.909	−202760 ii	1874470 ll
succinyl thioester	−496.6 n	394.5 h	140.5 h	216.1 h	8.7769	15562.9 ff	115.513	−180666	113.931	496645 jj	−13856 mm
succinyl thioester−1	−468.9 o	292.0 h	133.7 h	78.0 h	8.4641	16436.3 ff	−17.979	−184277	133.088	−162424 ii	187278 ll
acetyl thioester	−140.1 n	400.1 h	107.3 h	255.5 h	6.9314	10305.7 gg	36.887	−158933	171.102	342400 kk	−160625 mm

a-kJ mol−1.

b- J mol−1 K−1.

c-cm3 mol−1.

d-J mol−1 bar−1.

e- J mol−1.

f-J K mol−1.

g- J mol−1 K−1.

h-value as described in Table 1.

i- from [33].

j-calculated from pKa values from [33] and of oxaloacetate−2 from above.

k-from [38].

l-from [37].

m-from [41].

n-estimated as described in Figure 2.

o-estimated as was succinyl thioester in Figure 2, except using pentanoate, taken from [23], as the base structure.

p- estimated as was oxaloacetic acid in Figure 2, except using H-malonate−1, taken from [23], as the base structure.

q- estimated as was oxaloacetic acid in Figure 2, except using malonate−2, taken from [23], as the base structure.

r-calculated from of malic acid in above table using the Δ from [33].

s-estimated from of a-ketoglutaric acid from above table assuming the same Δ as between succinic acid and its respective ions in [23].

t-estimated from V0 of pyruvic acid in above table assuming the same Δ as between lactic acid and lactate, in [23].

u-estimated as was oxaloacetic acid in Figure 2, but using H-malonate−1, from [23] as the base structure.

v- estimated as was oxaloacetic acid in Figure 2, but using malonate−2, from [23] as the base structure.

w-from [42].

x-estimated by assuming the same Δ as between succinic acid, taken from [35] and its respective ions, taken from [23].

y-from [43].

z-from [36].

aa-from [44].

bb-estimated from of pyruvic acid in above table assuming the same as between lactic acid and lactate in [23].

cc-estimated as described in Figure 2 for oxaloacetic acid but using respective of H-malonate−1 or malonate−2.

dd-estimated form the acid in the above table assuming the same as between succinic acid and the respective −1 or −2 ion from [23].

ee – estimated using Equation (6)

ff-estimated using Equation (7).

gg- estimated using Equation (8).

hh-calculated from the a parameter in above table as described by [23].

ii- calculated using Equation (10).

jj- calculated using Equation (11).

kk- calculated using Equation (12).

ll-calculated from in above table using Equations (30–32) from [18].

mm-estimated as described in text.

The estimation of high temperature and pressure thermochemical properties for cis-aconitate and isocitrate, for the reactions between citrate and α-ketoglutarate, were not calculated due to deficient reference state data. Since citric acid and its ions are the only feasible tricarboxylated base structures available to use for the estimation, this approach was rejected to avoid circularity. Furthermore, the primary goal of estimating the high pressure and temperature parameters for CAC reactions calculated herein is primarily driven to examine these reactions under the near equilibrium conditions that are present in anoxic systems. Extant microbes that use the CAC reductively to fix carbon generally only contain a partial cycle where the pathway is used to supply precursors for biosynthesis via succinyl-Coenzyme A or α-ketoglutarate [2-4,45].

Calculation of the reference state entropy (S0 aq) and estimation of missing values

The S0aq values selected are shown in Table 1. If S0aq values were not available, third-law entropies were calculated from the aqueous enthalpy and Gibbs free energy values along with the sum of entropies of the elemental constituents using Equation (1):

The S0aq for some anions in Table 1 were calculated from the S0aq of the acid using Δ for the ionization reactions from Miller and Smith-Magowan [33]:

The aqueous formation entropies for H-α-ketoglutarte−1 and α-ketoglutarte−2 were estimated by assuming similar Δ from succinic acid and its mono- and divalent anions (from Shock [23]). The carbonyl group S0aq contribution value (54.3 J mol−1) used for oxaloacetic acid and its respective anions was calculated from the difference in S0aq between α-ketoglutaric acid and succinic acid from Shock [23]:

To calculate the S0aq needed for acetyl thioester the value of S0aq (-S-) was calculated from the difference in aqueous entropies between ethyl sulfide (see Appendix A, Equation 21), and n-butane [18].

The S0aq (>C=O) value used for succinyl thioester and its anion was estimated from the difference (68.15 J mol−1 K−1) in y-intercepts between n-alkanones and (n-1) n-alkanes, both from Shock and Helgeson [18]. The S0aq (>C=O) value used here was chosen since the value derived from an n-alkanone may be more likely to represent the value of a subterminal carbonyl adjacent to a sulfur atom, as opposed to the α-carbonyl adjacent to a carboxylic acid, as was used above for oxaloacetic acid.

Calculation of the reference state partial molal volume (V°aq) and estimation of missing values

The chosen partial molal volume values are shown in Table 1. The group value V0 (>C=O) used for the oxaloacetate series was estimated by the difference in V0 between α-ketoglutaric acid [36] and succinic acid [35] such that:

This V0 (>C=O) was used for pyruvic acid and pyruvate as well since it may rather well represent the volume of an α-carbonyl (V0 ~5 cm3 mol−1) adjacent to a carboxylic acid, rather than n-alkanone’s carbonyl with a much larger volume of (~14–15 cm3 mol−1 [30,31]).

The V0 for fumarate−2 was used to estimate the V0 of fumaric acid and the H-fumarate−1 anion by assuming the same Δ relationship for fumaric acid and its ions as for succinic acid from Criss and Wood [35], and H-succinate−1 or succinate−2 anion, from Shock [23], as demonstrated in Figure 2. The same relationship, using the Δ (13.13 cm3 mol−1) between lactic acid and lactate from Shock [23] was used to estimate the V0 for pyruvate from its acid.

The partial molal volume of the (-S-) group for a thioester was estimated by the addition of the value of V0 (-S-) from a value published by Lepori and Gianni [40] to the V0 of 2-butanone taken from Shock and Helgeson [18]. The V0 of succinyl thioester and its anion were estimated by the addition of V0 (-S-) [40] and V0 (>C=O) to the V0 of pentanoic acid and pentanoate, respectively (both from Shock [23]). The V0 (>C=O) value was estimated to be the difference between the V0 of n-alkanones and (n-1) n-alkanes (both taken from Shock and Helgeson [18]).

Calculation of the reference state heat capacity (C°p aq) and estimation of missing values

The standard molal isobaric heat capacities at the reference temperature and pressure are shown in Table 1 and estimation procedures in Figure 2. The of pyruvate ion was assumed to have the same difference in from its acid, as did the lactic acid-lactate pair [23], of 167.4 J mol−1 K−1. The for oxaloacetic acid was estimated by adding the value (−52.0 J mol−1 K−1) from that reported by Cabani et al. [31] to that of malonic acid [23]. For the estimation of values for both H-oxaloacetate−1 and oxaloacetate−2 ions, it was assumed that the difference from the acid was the same as between succinic acid and it respective anions from Shock [23]. This assumption was also used to estimate values of the H-mono- and divalent anions of malate, fumarate, and α-ketoglutarate. The of fumaric acid was estimated through subtracting the values for the difference (70.7 J mol−1 K−1) in y-intercepts between n-alkanes from n-alkenes (both values being from Shock and Helgeson [18]) from that of succinic acid. For α-ketoglutaric acid, the value (−52.0 J mol−1 K−1) reported by Cabani et al. [31] was added to that of succninc acid. The for succinyl thioester was estimated from addition of the , and the (−81.2 J mol−1 K−1), both calculated in this work, to the of pentanoic acid from Shock [23]. The for succinyl thioester was calculated from the difference in y-intercepts of C2,C4,C6 n-diaklysulfides [34] and C2,C4,C6 n-alkanes [23]. The value (−135.2 J mol−1 K−1) was calculated to be the difference between 2-pentanone and n-butane, both from Shock and Helgeson [18]. This value for was chosen for succinyl thioester because a carbonyl from an n-alkanone may be more likely to represent the in a thioester, as opposed to that from a α-carbonyl adjacent to a carboxylic acid. The resulting value of 216.1 J mol−1 K−1 is in close agreement with the sum of group values provided by Cabani et al. [31], using the (-S-) calculated here, of 216.4 J mol−1 K−1. The of the succinyl thioester anion was assumed to have the same difference from its acid as that between pentanoic acid and pentanoate [23]. The of acetyl thioester was estimated by adddition , as described above, to that of 2-butanone from Shock and Helgeson [18].

Extrapolation of reference state data to high pressures and temperatures

Starting with 25°C, 0.1 MPa reference state data, calculating the at the elevated P and T involves the integration of Equation (5) as:

The revised-HKF model allows for the incorporation of the changes in the partial molal volume and heat capacity as described in Appendix B.

Estimation of the temperature and pressure effects on the partial molal volume of aqueous organic species: the non-solvation contribution

The partial molal volume of a substance in the revised HKF model is defined by Equation (24). At temperatures of ≤ ~150°C, at the water-saturation vapor pressure (Psat), the solute-dependent contribution (the non-solvation volume, ) to the partial molal volume term dominates the partial molal volume. The term is calculated from Equation (25) utilizing fitting parameters (a1, a2, a3, and a4) to integrate the term into V0 at a desired pressure and temperature. The a1 and a parameters have been generated from empirical data gathered at high pressure and temperature conditions for a variety of compounds [11,15-20,46,47]. The a1 variable is correlated to a high degree with the of a wide range of neutral and charged aqueous organic compounds with a variety of functional groups (Figure 3) and can therefore be used to estimate the values of a1 for compounds with structural homology to those with high pressure and temperature data for which there are no volumetric data beyond the reference state.

Figure 3.

Regression plot of the non-solvation parameter a against the non-solvation volumes of short-chained aqueous organic species taken from the literature [15,18,23]. The non-solvation volumes were calculated using Equation (25) with the partial molal volumes and effective Born coefficients of (C3-C5) carboxylic acids, (C2-C6) carboxylate anions, (C3-C5) hydroxy acids, (C3-C6) hydroxylate anions, (C2-C6) dicarboxy acids, dicarboxylate−1, and dicarboxylate−2 anions [23], (C3-C5) n-alkanones, n-alkanes, n-alkenes, n-alcohols [18] and (C3-C5) aldehydes [15].

For the range of compounds regressed in Figure 3, the line is defined by: where a1 is in J mol−1 K−1 and in cm3 mol−1. This regression equation was used to generate the a1 parameter for all neutral and charged compounds in this work.

The a2 parameter is somewhat more dependent on the functional group characteristics of a particular molecule. In Figure 4 the of aldehydes, hydroxy acids, carboxylic acids, and dicarboxylic acids, and their respective anions, are plotted against a2.

Figure 4.

Regression plot of the non-solvation parameter a against the non-solvation volumes of short-chained aqueous organic species taken from the literature [15,18,23]. (a) Regression of non-solvation volumes calculated with Equation (25) using the partial molal volumes and effective Born coefficients of (C3–C5) carboxylic acids, (C2–C6) carboxylate anions, (C3–C5) hydroxy acids, (C3–C6) hydroxylate anions, (C2–C6) dicarboxy acids, −1, and −2 anions [23], and aldehydes [15]. (b) Upper line: Regression plot generated from points in upper figure. Lower line: Regression of non-solvation volumes calculated with Equation (25) using the partial molal volumes and effective Born coefficients of (C3–C5) n-alkanones, and (C2–C5) n-alcohols [23].

The data are fit by the line where a2 is also in J mol−1 K−1. For n-alkanones and n-alcohols (Figure 4) the slope is somewhat shallower and the y-intercept lower so these values are fit better by the line

The a2 parameter for all dicarboxylic acids, and dicarboxylate anions, citric acid and its anions, pyruvic acid and pyruvate, and succinyl thioester and its anion were estimated with Equation (7). Since the only apparently large departures from the line in Equation (7) are for shorter- chained carboxylate and hydroxylate anions, a fit for hydroxylates was considered separately to calculate the a2 parameter for pyruvate. The y-intercept for hydroxylates (Equation (9)) is slightly lower with a steeper slope than Equation (8):

However, if the y-intercept value for n-alcohols vs. n-alkanones can be considered comparable to that of the hydroxy and carbonyl groups in hydroxy- and α keto-acids, respectively, then pyruvate’s y-intercept would shift toward the line in Equation (7). Therefore the a2 value for pyruvic acid using Equation (7) was retained. The a2 parameter for acetyl thioester was estimated with the line defined by Equation (8) for n-alcohols and n-alkanones.

All a4 parameters were generated, as suggested by Shock and Helgeson and Shock [18,23], using the correlation with the a2 fitting parameter. The a3 parameter was then calculated by solving the rearranged non-solvation molal volume term ( ) of Equation (25) at 25 °C and 0.1 MPa.

Estimation of temperature and pressure effects on the isobaric heat capacity of aqueous organic species: The non-solvation contribution

The non-solvation and solvation heat capacity contributions to the partial molal heat capacity are defined in Equation (27). The non-solvation contribution term of Equation (27) is expanded in Equation (28) and combines the influence of pressure on the with incorporation of a substance’s a and a fitting parameters, from above, and the influence of temperature by the use of two heat capacity fitting parameters, c and c. The c parameter correlates closely with the reference state for compounds with similar functional groups (Figure 5).

Figure 5.

Regression plots of the non-solvation molal heat capacity variable c2 against the reference state heat capacity of neutral and ionic organic species from the literature [18,23]. (a) Plot of organic acid anions: (C2–C5) hydroxylates, (C2–C5) carboxylates and (C3–C6) H-dicarboxylate−1 and dicarboxylate−2 ions (all from reference [23]) used to generate Equation (10). (b) Upper line: Plot of neutral acids: (C4–C5) carboxylic acids, (C3–C5) hydroxy acids, and (C4–C6) dicarboxylic acids from reference [23] used to generate Equation (11). Lower line: Plot of (C3–C5) n-alkanones and (C2–C5) n-alcohols from reference [18] used to generate Equation (12).

Therefore, from this correlation high temperature and pressure data from structurally similar compounds can be used to predict the c parameter of molecules for which no high temperature heat capacity data are available. Figure 5a shows the plot of c2 vs. values, taken from Shock [23], for organic acid anions. The regression of these values for the range of compounds shown is described by the line in Equation (10): where c2 is in J mol−1. This regression equation was used to estimate the c2 parameters for all the dicarboxylate and carboxylate anions from the reference state used in this study. Equation (11) describes the upper line in the plot of c2 vs. values from organic acids (taken from Shock [23]) in Figure 5b: which was used to estimate the c2 parameter for all organic acids. The lower line in Figure 5(b) is a plot of the c2 vs. of short-chained n-alcohols and n-alkanones [18] and is described by the line in Equation (12), which was used to estimate the c2 parameter for acetyl thioester:

Estimation of temperature and pressure effects on the partial molal properties of aqueous organic species: The solvation contribution

At temperatures of ≥~150°C, at the water-saturation vapor pressure (Psat), the solvent-dependent contribution to the partial molal volume (Equation (26)) and heat capacity (Equation (29)) terms begin to dominate each partial molal function. The conventional (ω) and effective (ωe) Born coefficients, respectively, are used to describe the substance-specific solvation properties of an ionic species or electrolytes, and neutral species (see Appendix B).

Calculation of the conventional Born coefficient (ω) for ionic species

The ω of ionic species has been demonstrated to have a strong correlation with the [17] and can therefore be calculated from this quantity. In Figure 6 ω is plotted against the for a variety of mono-, di- and trivalent inorganic and organic anions (taken from Shock and Helgeson (1988) [17]) using relationships in Equations (30–32) to calculate ω, which was also used to calculate the ω for all anions in this work (data also shown in Figure 6).

Figure 6.

Regression plot of the conventional Born coefficients against the partial molal entropy of various anions. The upper, middle, and lower lines are the correlations for the tri-, di-, and monovalent anions, respectively, from Equations (30–32) using values of inorganic ions (taken from Shock and Helgeson [17]), organic anions (taken from Shock [23]) and compounds calculated in this work.

Calculation of the effective Born coefficient (ωe) for neutral species

The effective Born coefficient used to describe the solvation contribution of neutral species in the revised-HKF model has been calculated from [13,22,23] and the Gibbs free energy of hydration (Δ0) (Appendix C) in the absence of high temperature and V0 data. It has been demonstrated from empirical data that ωe generally has a negative value for low- molecular weight neutral organic compounds. The negative value is the result of the inflection of and V0 values towards positive-infinity near the critical point of water. As the properties of a set of molecules become increasingly polar, the solute-solvent interaction increases. In the case of some neutral inorganic polyhydroxyl compounds such as aqueous silica, of which the hydrated form is thought to be H4SiO4, and boric acid (H3BO3), both demonstrate electrolyte-like behavior and thus positive ωe values. The mechanism associated with this phenomenon is thought to arise from water-solute versus water-water competition near the critical point [48], where solutes that are associated with more solvent molecules than is the solvent itself, and will have and V0 values that approach −∞. The volatility of a substance in comparison to water is also thought to have influence over this process as well. This issue is discussed in detail by Amend and Plyasunov [49], where predictions were made concerning the near-critical point behavior of carbohydrates. It is clear, through their discussion and others, that the relationship between ω and solvation at higher temperatures is not an obvious one. For instance, the non-electrolyte amino acid proline displays low-temperature solvation behavior that is best described with a negative value for ωe. As the temperature increases, however, proline’s solvation is best fit with a positive ωe. However, this type of solvation behavior may be particular to the proline zwitterion due to its particularly asymmetrical dipole and spatial arrangement of hydrophobic and hydrophilic moieties [22]. If the high density of polar functional groups, as in polyhydroxy compounds, is to be viewed as an indicator of near-critical behavior of compounds with hydroxyl functionalities, then for dicarboxylic acids, oxalic acid with an ωe that displays neutral behavior may be the closest analogue.

In Table 1, different values for ω are displayed as calculated from the correlations with the Δ0 (Equation (44)) and entropy (Equation (45)), as has been done previously. Also included is a systematic estimation of ωe using values provided by Shock and Shock and Helgeson [18,23] with results shown in Figure 7 in comparison to other series of neutral organic compounds.

Figure 7.

Plot of the effective Born coefficient vs. the number of carbon atoms of neutral organic compounds. The lines are regressions generated from values of the selected functional series of compounds noted in Table 1. Datum points are the values of ω for the neutral compounds calculated as described in the text.

The values of these estimations were used for ωe in all the neutral compounds in this work. The ωe for pyruvic acid was estimated by taking the ωe from lactic acid and subtracting the difference between n-propanol and acetone:

The ωe for oxaloacetic acid was estimated from the value for malonic acid with addition of the carbonyl group value such that:

Malic acid’s ωe value was estimated by from succinic acid from: to add the value of the hydroxyl group. For fumaric acid the difference in n-alkanes and n-alkenes were used to modify succinic acid as:

The ωe for α-ketoglutaric acid also used succinic acid and carbonyl value for oxaloacetic acid:

Since there are currently no analogous compounds (tricarboxyl) for citric acid, its ωe was estimated to be the same as a C6 compound using the slope and y-intercept for dicarboxylic acids. The value for succinyl thioester was estimated by: using the value from [22] for diethyl sulfide. The ωe estimation for acetyl thioester also used the same value for diethyl sulfide:

In consideration of consistency with previous works and the pragmatic aspects of the temperature ranges to be expected to be relevant for these compounds, negative values for ωe were chosen. In addition, for practical purposes, since the pKa of the strongest acid among these compounds is 2.49 (pyruvic acid from reference [33]), at pH above 3–4 the acids of these compounds will not usually be germane in writing reactions.

Results

Analysis of the possible error associated with methods used in estimating reference and standard state parameters

Since the motivation for this work is to estimate the high temperature and pressure thermochemical parameters for compounds for which there was no data, the credibility of the resulting values need be examined. The effects of errors on the reference state data, as well as for values calculated beyond the reference state, can be analyzed by the comparison of expected calculations, using the estimated values, with calculations from values in which a sensible error is incorporated. Figure 8a demonstrates the influence of the over- and underestimation of ω on the partial molal volume of propanoic acid along Psat calculated using Equations (25) and (26) with revised HKF parameters from Shock [23]. Although the reference state is unaffected by ωe, the error in the calculated increases with temperature as the partial molal volume is influenced increasingly by the solvation term, although at higher pressures the error is diminished (Figure 8b). However, as we are most concerned with providing data for reaction thermodynamics at elevated pressure and temperature, the effects of inaccurate estimation of the are of primary concern. As can be seen in Figure 8c, the revised-HKF method is quite insensitive to even a 2-fold under- or over estimation of ωe, with a maximum of ~0.3% relative error at 350°C. The insensitivity to errors from the estimation methods can be further tested by swapping the HKF parameters of one compound for another, while using the original . To demonstrate this, for compounds in Figure 8d the original values were retained while the remaining values (S0aq, a–a, c, c, and Born coefficients) were mutually swapped from another compound: pyruvic acid and propanoic acid, H-oxaloacetate−1 and H-α-ketoglutarate−1, and oxaloacetate−2 and α-ketoglutarate−2 (all values are from Table 2 or Shock [23] for propanoic acid) and used to solve Equation (39). The largest error (~4% relative at 350 °C) is encountered when the equation of state parameters from propanoic acid are replaced with those of pyruvic acid. Considering that there is a relative difference (compared to pyruvic acid) of 15%, 24%, and 12% between the S0aq, V0, and of propanoic acid, respectively, this level of error is quite tolerable, as group additivity estimations generally give relative errors of ~5% for these classes of organic compounds [50]. At 150°C the error is approximately half that at 350°C and is likely to fall below the analytical errors encountered when quantifying the concentrations of these substances.

Figure 8.

Plot of the error in partial molal properties of neutral and ionic organic compounds expected from the improper estimation of HKF parameters as a function of temperature and pressure. (a) and (b) Partial molal volume of propanoic acid, taken from [23], calculated using Equations ((25) + (26)) with the over- and under-estimation of the effective Born coefficient. (a) The solid line is the predicted V0 of propanoic acid at Psat using the ω from [23]. The upper dashed-line is the V0 predicted by underestimating ω by 0.5-fold. The lower dashed-line is the V0 predicted by a 2-fold overestimating of ω. (b) The percent relative error expected in V0 as a function of pressure at 0.5x and 2.0x ω. (c) The effect of the over- and under-estimation of ω on relative error in ΔG at Psat (from Equations (38) and (39)). (d) Plot demonstrating the relative error in ΔG at Psat expected from gross misestimation of HKF parameters. Using the reference state (from Table 1) for the labeled acid or ion, the remaining values ( , a, a, a, a, c, c, and ω) were swapped: propanoic acid (taken from [23] for pyruvic acid, and the respective α-ketoglutarate anion for H-oxaloacetate−1 and oxaloacetate−2 ions (Table 1), and vice versa.

Table 2.

The effective Born functions (ω J mol−1) used for neutral organic species calculated by different methods.

	ωa	ωb	ωc
pyruvic acid	−1.2922	−1.2679	1.2156
oxalaoacetic acid	−0.5216	−0.4495	2.4908
malic acid	−0.7535	−0.5100	2.7785
fumaric acid	−0.9565	−0.7306	−1.7418
a-ketoglutaric acid	−0.3512	−0.2814	1.3690
citric acid	−0.2333	−0.2780	4.0067
succinnyl thioester	−0.1386	0.0107	7.7939
acetyl thioester	−1.6063	0.0453	−3.7706

a-calculated as described in text and Figure 6.

b-calculated from aqueous entropies from Table 4 using Equation (45).

c-calculated from the hydration Gibbs free energy using Equation (44).

Estimation of equilibrium constants at high temperatures and pressures

As an example of the usefulness of these data, values of the logarithm of the equilibrium constant (log K) for acid dissociation reactions were calculated at different temperatures from the values at Psat using Equations (38) and (39) and: with the partial molal properties and equation of state parameters in Table 1 (Figure 9). These values allow us to evaluate the potential for each of these reactions to occur under different geochemical conditions. The plots in Figure 8 allow investigation of the pH dependence of the speciation among CAC components. Similarly, the data and parameters, along with the revised HKF equation of state, allow evaluation at wide ranges of temperatures and pressures of reaction energetics among species in the various steps of the CAC (see Figure 1) to determine the thermodynamic viability of these reactions for a variety of conditions. If life did indeed begin under hydrothermal geochemical conditions, these calculations can help identify the conditions necessary for this development.

Figure 9.

Plot of the logarithms of equilibrium constants for dissociation reactions (as indicated) involving organic species from this work as a function of temperature at Psat.

Concluding Remarks

Using the thermodynamic data that have either been measured experimentally or estimated through methods described and provided in this paper for the constituents of the citric acid cycle, we can begin to place the fundamental biological process of energy transfer into a geochemical context. With the data and parameters presented in this paper, we can for the first time calculate thermodynamic reaction properties for the citric acid cycle under hydrothermal conditions, whence life may have emerged. Furthermore, we can use calculations such as the ones described above to evaluate the energy cycles of microorganisms that live at elevated temperatures and pressures and thus gain insight into the conditions necessary for the initiation of these cycles. We are now also able to evaluate quantitatively the steps in the reverse or reductive citric acid cycle, which may have preceded the more modern oxidative citric acid cycle as the primary energy transfer mechanism for life. Such calculations are facilitated through use of the computer program SUPCRT92 [51].

In addition, we can gain some insight into the conditions (including factors such as pH, temperature, pressure and concentrations of the chemical components) on the early Earth that may have facilitated the initiation of the central metabolic pathways such as the reductive and oxidative citric acid cycles in biological energy systems by evaluating quantitatively the energy gained through various metabolic reactions.

For example, careful examination of Figure 9 reveals that the logarithms of the equilibrium constants of many of the deprotonation reactions involving components of the citric acid cycle vary by up to an order of magnitude over the known temperature range for life (currently up to 122°C). Because these reactions are functions of pH, changes in the equilibrium constants over the temperature range will change the range of pH at which they would be thermodynamically favorable. A full evaluation of the geochemical parameters attending any environment would be required to determine the effect each would have on reaction favorability and the viability of the citric acid cycle.