Hydrolysis of glucose-6-phosphate in aged, acid-forced hydrolysed nanomolar inorganic iron solutions—an inorganic biocatalyst?

Abstract

Phosphate ester hydrolysis is one of the most important chemical processes in biological systems. Although catalysis by the natural phosphoesterases, e.g., purple acid phosphatase (PAP) and its biomimetics, are well known in biochemistry, it has been reported that some metals and mineral phases can significantly facilitate the hydrolysis of phosphate ester. Here we report for the first time that aged, acid-forced hydrolysed nanomolar inorganic iron solutions significantly promoted the hydrolysis of glucose-6-phosphate (G6P), and that the reaction kinetics followed the Michaelis–Menten equation. The catalysis was inhibited by tetrahedral oxyanions in an order of WO₄ > MoO₄ > PO₄. The newly formed oxo-bridge or hydroxo-bridge during the iron-aging process might contribute to this biocatalytic effect, though the detailed mechanism is still unclear. Further studies are needed in order to understand the (hydr)oxo-bridged Fe–Fe structure in water and its role in organic phosphorus transformation. This catalyst might be one of many ubiquitous sets of inorganic enzymes yet to be discovered in nature that act as a bridge between the inorganic and organic worlds, and would have played a critical role in the origin of life.

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1. Introduction

Glucose-6-phosphate (G6P), an example of a phosphate ester, is glucose sugar phosphorylated on carbon 6, and widely distributed in nature, including water and soil.^1–3 As an essential process of life,^4–8G6P hydrolysis is catalyzed by various enzymes, including phosphoesterases^9–15 such as purple acid phosphatase (PAP). It is also known that their biomimetics, i.e., metals, especially iron, chelated to special organic ligands, can significantly promote the hydrolysis of phosphate ester.^16–28 Moreover, it has been reported that some metals^29–32 and mineral phases^33–37 can significantly facilitate the hydrolysis of ester phosphate, including nucleoside phosphates. Therefore, understanding the behavior of phosphate ester hydrolysis is important for biology and biogeochemistry as it is relevant to phosphate availability in the environment.^2,38–43

Fig. 1 Hydrolysis of G6P in a 16.5 nM Fe(NO₃)₃ solution aged for 14 months at room temperature (22 ± 2 °C). (a) Concentration of IP and G6P during 20 μM G6P hydrolysis. (b) Pseudo first-order reaction kinetics of G6P. (c) Double reciprocal (initial velocity and initial concentration of G6P) plot (Lineweaver–Burk plot) of G6P from 5 to 250 μM. (d) Initial velocity of G6P hydrolysis (v) as a function of the initial concentration of G6P.

Table 1 Hydrolysis rate constant of 20 μM G6P in aged inorganic iron solutions^a

Fe source	Manufacturer	Aging time (month)	Total Fe conc. (nM)	Rate constant (10^−6 s^−1)	Half-life (h)
a The hydrolysis rate constant of initial 20 μM G6P in DIW is 1.53 × 10^−7 s^−1, and the half-life is 1255 h.
Fe(NO3)3	J.T.Baker	14	16.5	30.17	6.4
	Riedel-de Haën	4	16.5	5.08	37.8
			100	2.96	65.6
		6	1000	18.77	10.3
Iron standard solution (metal Fe in 0.3 M HNO3)	J.T.Baker	4	1	2.38	81.0
			2.5	4.85	39.7
			7.5	7.22	26.7
			50	6.38	30.2
			100	6.55	29.4
			200	6.58	29.2
			500	8.14	23.6
			1000	5.86	32.8
FeCl3	J.T.Baker	16	2	6.59	29.2
			10	4.95	38.9
	Riedel-de Haën	4	10	3.28	58.6
			100	0.83	231.9
Fe(ClO4)3	Aldrich	4	10	2.46	78.4
			100	0.61	318.3
Fe(NH4)2(SO4)2	EM Science	16	16.5	9.49	20.3
Fe-EDTA	Self-made	16	16.5	4.24	45.4

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Recently, we discovered that hydrolysis of G6P is significantly promoted by aged, acid-forced hydrolysed nanomolar iron solutions, and the presence of some tetrahedral oxyanions in solution act as inhibitors in an order of WO₄ > MoO₄ > PO₄. The kinetics of G6P hydrolysis in these nanomolar inorganic iron solutions can be described by the Michaelis–Menten equation. Here, we report these interesting phenomena and hypothesize that the (hydr)oxo-bridged Fe–Fe structure in the aged iron solution contributes to the observed catalytic effect.

2. Experimental

Deionized water (DIW), used for preparing standards, reagents, and aged iron solutions, was purified first with a distilling unit and then by a Millipore Super-Q Plus water system that produced water with a resistivity of 18 MΩ cm. All reagents and aged iron solutions were stored in polypropylene bottles (or containers) that were immersed in a 10% HCl solution overnight, followed by rinsing three times with DIW and then drying at 60 °C in an oven for 5–10 h prior to their use.

All chemicals used were of analytical grade (AR or GR), and used as received. Glucose-6-phosphate (D(+)-glucopyranose 6-phosphate sodium salt, G-6-P Na, C₆H₁₂NaO₉P, F. W. 282.12), glycerol 2-phosphate (β-glycerophosphate disodium salt hydrate, G2P, C₃H₇Na₂O₆P, F. W. 216.04), ribose-5-phosphate (D-ribofuranose 5-phosphate disodium salt, R5P, C₅H₉Na₂O₈P·xH₂O, F. W. 274.07), and fructose 1-phosphate (D-fructose 1-phosphate sodium salt, F1P, C₆H₁₁O₉PNa₂, F. W. 304.10), were purchased from Sigma and stored in a freezer (−20 °C). A 50 mM stock solution for each organic phosphorus compound was made individually, stored in a refrigerator at 4 °C, and diluted to suitable concentrations for daily hydrolysis experiments.

Inorganic phosphate (IP) derived from the hydrolysis of G6P was determined by a modified method of Murphy and Riley in which the final total pH_T of the test solution (sample mixed with reagents) was 1.0 (H/Mo = 70).⁴⁴ An ammonium molybdate reagent was prepared by mixing 2.4 g of ammonium molybdate ((NH₄)₆MO₇O₂₄·4H₂O, Merck, GR), 25 mL of concentrated sulfuric acid (H₂SO₄, 96–98%, J.T. Baker), and 50 mL of 0.3% antimony potassium tartrate (K(SbO)C₄H₄O₆)₂·H₂O, Fisher) solution and diluting the mixture to 1 L with DIW. An ascorbic acid solution was prepared daily by dissolving 1 g of ascorbic acid (C₆H₈O₆, Aldrich, AR) in 100 mL of DIW. Prior to sample analysis, a colour reagent was prepared by mixing equal volumes of the molybdate reagent with the ascorbic acid solution. One ml of the colour reagent was then added to 4 ml of the sample and mixed. After 10 min, absorbance of phosphoantimonyl-molybdenum blue was measured at 890 nm with background corrections at 780 to 1020 nm by a Hewlett-Packard 8453 UV-visible spectrophotometer.

2.1. Preparation of aged iron salt solutions

Since acidic environments significantly reduce the rate of iron(III) hydrolysis,^45–48 six series of inorganic iron(III) salt solutions were made by rapid dilution under acidic conditions to nanomolar concentrations. Five different iron salts and a commercial iron standard solution for atomic absorption spectrophotometry (J.T.Baker, prepared by dissolving metal Fe in 0.3 M HNO₃ to yield a concentration of 1000 mg ml⁻¹) were used. The total iron concentration in the solutions used for G6P hydrolysis experiments ranged from 1 to 10 000 nM, and the maximum aging time was 16 months.

2.1.1. Aged 16.5 nM Fe solutions. A 10 mM Fe solution was prepared by dissolving 2.02 g of Fe(NO₃)₃·9H₂O (J.T.Baker, AR, F. W. 404.00) or 1.96 g of Fe(SO₄)₂(NH₄)₂·6H₂O (EM Science, GR, F. W. 392.14) in 500 ml of dilute HNO₃ solution (pH 3.0). This solution was immediately diluted 10 times with DIW. Such dilution (a 10 to 1 ratio) by DIW was repeated with newly prepared, an order of magnitude lower concentration of Fe solutions until the Fe concentration reached nM levels. All sequential dilutions were processed within 10 min to minimize Fe hydrolysis at higher concentrations. A 500 nM Fe-EDTA solution was also prepared by first diluting the above mentioned 10 mM Fe(NO₃)₃ 10 times with DIW and adding EDTA at a molar ratio of 1 : 4 (Fe : EDTA) to make a 1 mM Fe-EDTA solution and subsequent step-wise dilutions (10 times dilution at each step). After 3 months, a 50 nM Fe solution was made from the aged 500 nM Fe solution by dilution with DIW. After another 2 months of aging, 2 mM of NaHCO₃ was added to the 50 nM aged iron solution, to yield a final concentration of 16.5 nM iron in 0.67 mM NaHCO₃. The pH of the aged iron solution was adjusted to 6.3 ± 0.1 by adding either 0.1 M HCl or 0.1 M NaOH or 20 mM NaHCO₃ five times during the aging process. A noticeable catalytic reactivity was detected after 6 months of aging. The final measurement was made at either 14 or 16 months.

2.1.2. Aged 1000 and 10 000 nM Fe(NO₃)₃ solutions. A 100 mM Fe solution was prepared by dissolving 2.02 g of Fe(NO₃)₃·9H₂O (Riedel-de Haën, AR, F. W. 404.00) in a dilute HNO₃ solution (pH 3.0). This solution was then diluted with DIW (pH 6.2) step-by-step (10 times dilution at each step) to produce 1000 and 10 000 nM Fe solutions. The dilution process was completed within 10 min. The pH of these iron solutions was adjusted to 6.3 ± 0.1 after aging for 140 to 300 days. The hydrolysis rate of G6P was measured at 6 and 10 months. All inhibition experiments and buffer effects were made with this aged 1000 nM Fe solution.

2.1.3. Aged 1 to 5000 nM ferric solutions. This series of ferric solutions was prepared from J.T.Baker's iron standard solution (1000 mg ml⁻¹Fe(NO₃)₃). A 1 mM Fe(III) solution was made by diluting the stock Fe solution with diluted HNO₃ (pH 3.0). A 5 μM solution was made by step-wise dilution (2–10 × dilution at each step by DIW) from the 1 mM Fe solution. A series of 50–5000 nM Fe solutions were prepared by diluting the 5 μM Fe solution. A series of 1–10 nM Fe solutions were prepared by diluting the 50 nM Fe solution. These dilutions were completed within 10 min. The pH of these Fe solutions was maintained at 6.3 ± 0.1 by adding dilute HCl or NaOH.

2.1.4. Aged 2 and 10 nM FeCl₃ solutions. A 10 mM FeCl₃ solution was prepared by dissolving 0.811 g of FeCl₃ (J.T.Baker, AR, F. W. 162.20) in 500 ml dilute HCl (pH 3.0), and then diluting it with DIW (pH 6.20) step-by-step (10 × dilution at each step) to a 500 nM Fe solution (within 3 min). After aging for 6 months, 2 and 10 nM Fe solutions were made from the aged 500 nM Fe solution, and the pH of the aged iron solutions was adjusted to 6.3 ± 0.1.

2.1.5. Aged 10 and 100 nM FeCl₃ solutions. A 10 mM FeCl₃ solution was prepared by dissolving 0.811 g FeCl₃ (Riedel-de Haën, AR, F. W. 162.20) in 500 ml dilute HCl (pH 5.0), which was then diluted step-by-step by DIW (pH 6.20) to make a final concentration of 10 and 100 nM (procedure described in Section 2.1.2). The pH of these nM iron solutions was adjusted to 6.3 ± 0.1 after 100 days.

2.1.6. Aged 10 and 100 nM Fe(ClO₄)₃ solutions. A 10 mM Fe(ClO₄)₃ solution was prepared by dissolving 1.77 g Fe(ClO₄)₃·xH₂O (Aldrich, GR, low chloride, Cl < 0.005%, F. W. 354.2) in 500 ml dilute HCl (pH 5.0), and then diluting it step-by-step (10 × dilution at each step) by DIW (pH 6.2) to make a final concentration of 10 and 100 nM (procedure described in Section 2.1.2). The pH of these iron solutions was adjusted to 6.3 ± 0.1 after 100 days.

2.2. Measurement of hydrolysis rate of G6P

The increase in concentration of IP due to hydrolysis of G6P was measured at suitable time intervals with a modified colorimetric method after G6P was added to the aged iron solution. The concentration of G6P at a given time, t, was calculated using [G6P]_t = [G6P]₀ − [IP]_t, where [G6P]₀ is an initial concentration of G6P and [G6P]_t and [IP]_t are the concentrations of G6P and IP at time t, respectively. A small amount of IP was initially present in the G6P as an impurity, and was subtracted as a blank. The first-order hydrolysis rate constant, k (s⁻¹), was determined from the slope of the linear portion of a log[G6P]_tvs. time (s) plot. The typical correlation coefficient of the linear fitting was 0.99*; the duration of the hydrolysis experiments ranged from 24 to 48 h (with the exception of the control). The time courses of formation of phosphorantimonylmolybdenum blue complex from phosphate released from hydrolysis of 20 μM G6P in an aged (4 months) 1000 nM iron solution at room temperature (22 ± 2 °C) at different hydrolysis time (0, 1, 3, and 6 h) is presented in the ESI Fig. 1.†

In the early stages of these experiments, either azide or CHCl₃ was added to the aged iron solution to test for potential microbial contamination. No significant difference was observed between inhibitor-added and no-inhibitor-added experiments. In the subsequent experiments reported here, none of the microbial inhibitors were used.

2.3. Inhibition experiments with different tetrahedral oxyanions

All inhibition experiments were conducted with the 10 month aged 1000 nM Fe(NO₃)₃ solution. The tetrahedral oxyanions included orthophosphate (PO₄), molybdate (MoO₄) and tungstate (WO₄), and were added as a stock solution into the aged iron solution before the G6P solutions were introduced. IP concentration was measured at different times and the hydrolysis rate constant was calculated as described in the previous section.

3. Results and discussion

3.1. Promotion effect of sugar phosphate hydrolysis in aged iron solutions

G6P hydrolysis without enzymes is a slow process in DIW and fresh nanomolar inorganic iron solutions. The concentration of IP in the solution containing 20 μM G6P in pure water (DIW, pH 6.2) was 0.42 ± 0.02 μM initially (t = 0), and increased slowly with time. The IP concentration was 0.59 ± 0.08 μM at 24 h, and reached up to 2.4 μM after 9 days at room temperature (22 ± 2 °C). The IP concentrations in the fresh nanomole inorganic iron solutions (0.5 to 50 nM Fe(NO₃)₃) was 0.82 ± 0.11 μM after 24 h. However, after G6P was added into the aged nanomolar inorganic iron salt solutions, made by acid-forced hydrolysis, the IP was rapidly released. For example, after an initial 20 μM of G6P was added into a 14 month aged 16.5 nM Fe(NO₃)₃ solution (pH=6.3) at room temperature, IP was rapidly increased due to the hydrolysis of G6P. The concentration of IP at 1, 3 and 6.7 h was 2.8, 6.4 and 10.6 μM, respectively. Therefore, the hydrolysis of G6P was significantly promoted in the aged inorganic iron solution (Fig. 1a). Like metal ions as well as natural and biomimetic enzymes, the kinetics of G6P hydrolysis in the aged iron solution can be described as the pseudo-first-order reaction (Fig. 1b).^{22,23,29,30,32,49–51} In this experiment, the decrease in G6P concentration, [G6P]_t, due to its hydrolysis can be expressed as a function of hydrolysis time, t, as

log[G6P]_t = −0.0000131t − 4.718 (r² = 0.999) (1)

where [G6P]_t is in M and t is in seconds.

The corresponding reaction rate constant (k) was 3.02 × 10⁻⁵ s⁻¹, and the half-life (t_1/2) was 6.38 h. In contrast, the average reaction rate constant in DIW and these fresh unaged nanomolar inorganic iron solutions was 1.53 × 10⁻⁷ and 1.98 × 10⁻⁷ s⁻¹, respectively, and the half-life was 1255 and 1100 h, respectively. It is noted that the hydrolysis rate constant in the aged iron solution was much higher than previously reported rates in the presence of the millimole metals.^29–32

The first-order reaction kinetics predicts a constant half-life of reaction regardless of the initial reactant concentrations. Consequently, the initial hydrolysis velocity of G6P (v_o) is the product of the hydrolysis rate constant (k) multiplying the initial concentration of G6P ([G6P]₀). However, the half-lifes and the hydrolysis rate constants at different initial G6P concentrations were not constant. For G6P hydrolysis at an initial concentration of 100 μM, the reaction rate constant was 8.83 × 10⁻⁶ s⁻¹, and the half-life was 21.8 h. This suggests that there was some intermediate product formation related to the initial concentration of G6P and a constant equilibrium of intermediate products might be established during the hydrolysis process, after the initial G6P was introduced into the aged iron solution. Similar to the general chemical process of catalysis, inorganic phosphate was released and the activated iron species were regenerated. It was observed that the initial velocity of G6P hydrolysis in the aged iron solution can be described by the Michaelis–Menten equation, a typical behavior of biocatalysis, in a range from 5 to 250 μM (Fig. 1c, and 1d).

where [G6P]_o is in M and v_o is in M s⁻¹.

The maximum initial velocity of G6P hydrolysis was about 1 nM s⁻¹, or 3.6 μM h⁻¹, and the Michaelis–Menten constant (K_m) was 13.7 μM. This is in strong contrast to previously reported promotion effects by metals^29–32 and minerals.^33–37

The promotion effect of G6P hydrolysis can be extended to 2500 μM in this aged iron solution with a k of 6.53 × 10⁻⁷ s⁻¹, and t_1/2 of 295 h. It should be pointed out that the concentration of phosphorus in the solution was 10³–10⁵ higher than that of iron (e.g., 16.5 nM Fe and 2500 μM G6P).

The measured hydrolysis rate constants of the initial 20 μM G6P with different sources of iron, made by acid-forced hydrolysis, are listed in Table 1. There were some differences in the hydrolysis rate constants among the different salts. The half-life for 4 month aged Fe(NO₃)₃ (16.5 nM), FeCl₃ (10 nM) and Fe(ClO₄)₃ (10 nM) was 37.8, 58.6 and 78.4 h, respectively, though they were in the same order of magnitude. The nitrate ion, which is found in ferric nitrate, is also an oxidising agent though it has been suggested that nitrate exerts little, if any, effect on the hydrolytic polymerization of ferric ion.^52,53 Both ferric and ferrous salts have a promoting effect, although the ferrous ions would have been partially oxidized to ferric ion during the aging process.⁵⁴

These aged iron solutions also catalyzed the hydrolysis of other sugar phosphates, including glycerol 2-phosphate (3-carbon, G2P), ribose-5-phosphate (5-carbon, R5P), and fructose 1-phosphate (6-carbon, F1P) (Table 2). Preliminary results show that the hydrolysis rate constants at a given sugar phosphate concentration are the same order of magnitude among these different sugar phosphates. It is also noted that the rates of 5-carbon and 3-carbon sugar phosphates (R5P and G2P) are higher than those of 6-carbon sugar phosphate (G6P and F1P). As expected, the promotion effect was also found for the other phosphorus ester compounds, including the energy metabolism compounds (AMP, ADP and ATP, and even polyphosphate and pyrophosphate) as well as the RNA model compound (4-nitrophenyl phosphate ester). However, no promotion effects were observed for the hydrolysis of phosphonates (C–P bonded compounds, e.g., 2-aminoethylphosphonic acid, phosphono-formic acid) and inositol hexakisphosphate (IP6) (data not shown).

Table 2 Sugar phosphate hydrolysis in aged inorganic iron solutions

Sugar phosphate	Aged Fe solution	Initial OP (μM)	Rate constant (10^−6 s^−1)	Half-life (h)
Glycerol-2-phosphate (G2P)	Fe standard solution, 7.5 nM, 4 mo.	10	12.69	15.2
		20	6.40	30.1
		50	3.45	55.8
		500	0.51	374.3
	Fe(NO3)3, 1000 nM, 6 mo.	20	30.12	6.4
	FeCl3, 2 nM, 16 mo.	20	5.29	36.4
	Fe(NH4)2(SO4)2, 16.5 nM, 16 mo.	20	11.44	16.8
Ribose-5-phosphate (R5P)	Fe standard solution, 7.5 nM, 4 mo.	10	13.92	13.8
		20	8.15	23.6
	Fe(NO3)3, 1000 nM, 6 mo.	20	25.19	7.6
	FeCl3, 2 nM, 16 mo.	20	7.24	26.6
	Fe(NH4)2(SO4)2, 16.5 nM, 16 mo.	20	16.16	11.9
Fuctose-1-phosphate (F1P)	Fe standard solution, 7.5 nM, 4 mo.	10	8.66	22.2
		20	5.25	36.4
	Fe(NO3)3, 1000 nM, 6 mo.	20	17.08	11.3
	FeCl3, 2 nM, 16 mo.	20	5.29	36.4
	Fe(NH4)2(SO4)2, 16.5 nM, 16 mo.	20	8.50	22.6

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Further experiments with a range of 1 to 10 000 nM ferric standard solution (pH ≈ 6.3) were carried out to investigate the contribution of iron concentration to the aging process. The hydrolysis rate constants of initial 20 μM G6P in all these nanomolar (1 to 1000 nM) aged iron solution increased with aging time at room temperature (22 ± 2 °C) (Fig. 2). After 4 months of aging, the hydrolysis reaction rate constant of initial 20 μM G6P in aged iron solutions increased from 0.15 to 10 (× 10⁻⁶ s⁻¹), and the half-life of G6P hydrolysis decreased from 2 months to ∼1 day (Fig. 2). It is noted that the time of aging needed to achieve an appreciable G6P hydrolysis rate constant was closely related to the total iron concentration. For iron concentrations less than 100 nM, it took less than a month; whereas for higher iron concentrations (200 to 1000 nM), it took more than two months. However, no direct correlations between the hydrolysis rate constants at the same initial concentration of G6P and the total iron concentrations were observed, which might imply that the catalytic characteristics of aged iron solution is very complex. No significant promotion effect was observed if the iron concentration was more than 1000 nM, even if the aging time was extended up to 14 months (data not shown).

Fig. 2 Hydrolysis rate constant (k) of 20 μM G6P as a function of aging time in solutions of different iron concentrations.

3.2. Inhibition effect of tetrahedral oxyanions on the hydrolysis of G6P

When the tetrahedral oxyanions were introduced into this aged iron solution, the hydrolysis of G6P was significantly inhibited (Fig. 3a). In the experiments with initial 20 μM G6P in the 10 month aged 1000 nM iron solution, addition of 10 μM PO₄ reduced the hydrolysis reaction rate constant to 60% of the control (without PO₄), and the half-life was 5.51 h. When the PO₄ concentration was increased to 40 μM, the rate constant was reduced to 40%, and the half-life was 10.34 h. In the presence of 1 μM MoO₄, the hydrolysis rate constant was reduced to 78%, and the corresponding half-life was 4.01 h. In the presence of 10 μM MoO₄, the hydrolysis rate constant was further reduced to 12%, and the corresponding half-life was 26.3 h. When the molybdate was increased to millimolar concentrations, no hydrolysis of G6P was observed even at a higher concentration of G6P (100 μM). The strongest inhibition effect was observed in the presence of WO₄. The rate constant was approximately 43% when 1 μM WO₄ was added into the aged iron solution; the corresponding half-life of G6P was 7.42 h. When the concentration of WO₄ was increased to 10 μM WO₄, the rate constant was further reduced to 3%, and the corresponding half-life was extended to 109.3 h. It was found that the percentage reduction in hydrolysis reaction rate constant (y) is a rational function of the tetrahedral oxyanion concentration (x in μM), which can be expressed as the following equation where the coefficient α is related to the strength of the inhibitor. In this experiment, α was 1.373, 0.493 and 0.0649 for tungstate, molybdate and orthophosphate, respectively, with the r² of fitted equation in a range of 0.97–0.99 (p < 0.0001) (Fig. 3b).

Fig. 3 Glucose-6-phosphate hydrolysis in a 10 month aged , 1000 nM Fe(NO₃)₃ solution at room temperature (22 ± 2 °C). (a) Kinetics of hydrolysis of G6P (20 μM initial) in the aged iron solution with no addition, and with addition of 1 μM MoO₄, 1 μM WO₄ and 10μM PO₄. (b) Percentage reduction of the hydrolysis rate constant (k) of initial 20 μM G6P as a function of the concentration of different tetrahedral oxyanions.

The inhibition effect was also closely related to the initial concentration of the G6P (Fig. 4), and the modes of tetrahedral oxyanions and G6P were competitive. The aged iron solutions might be bound to either the tetrahedral oxyanions (PO₄, MoO₄, and WO₄) or G6P to form two different intermediates, but cannot bind to both at any given moment (Fig. 5). The intermediates with G6P release IP, and the process of G6P hydrolysis is completed with the regeneration of the active binding sites on the aged iron species. The intermediates with the tetrahedral oxyanions prevent the binding of G6P, and also have a dissociation reaction to regenerate the active binding sites on the aged iron species.

Fig. 4 Inhibiting behavior of different tetrahedral oxyanions on the hydrolysis of glucose-6-phosphate in a 10 month aged, 1000 nM Fe(NO₃)₃ solution at room temperature (22 ± 2 °C). (a) Effect of initial concentration of G6P on the initial hydrolysis velocity of G6P. (b) Lineweaver–Burk plot of aged iron solutions in the absence and presence of tetrahedral oxyanions.

Fig. 5 Schematic diagram of the catalysis process of G6P hydrolysis in the presence of the tetrahedral anions in the aged inorganic iron solutions. E is the aged inorganic iron species; A is the active binding sites on the aged iron species. S is G6P and I is tetrahedral anions. ES and EI are the two intermediates.

Results indicate that the apparent affinity of G6P to the Fe–Fe bond in the aged iron solution (binding sites) might decrease due to the competition of the inhibitor (PO₄, MoO₄ and WO₄), while the maximum velocity of G6P hydrolysis (v_max) was still unchanged. The catalytic and inhibition behaviors of this aged iron solution in the presence of 5 to 125 μM G6P can be described by the Michaelis–Menten equation. From the linear relationship observed in the Lineweaver–Burk plot, the apparent Michaelis–Menten constant (K_mapp) can be calculated using the following:

where the [G6P]_o is the initial concentration of G6P (M), [I] is the concentration of inhibitor (M), K_m is Michaelis–Menten constant, and K_i is the inhibitor’s dissociation constant.

The Michaelis–Menten equations for G6P hydrolysis in the 10 month aged iron solution were as follows:

without any addition:

with 1μM MoO₄:

with 1 μM WO₄:

with 5 μM PO₄:

with 10 μM PO₄:

The K_m, the G6P concentration at which the hydrolysis velocity reached one-half of maximum velocity (v_max/2), was 2.7 μM G6P in this aged iron solution with no inhibitors added, and the K_mapp with the addition of 1 μM WO₄, MoO₄, and 5 μM PO₄ and 10 μM PO₄ was 46.2, 14.1, 11.1 and 17.1μM G6P, respectively. Therefore, the K_i of WO₄, MoO₄, and PO₄ was determined to be 0.06, 0.24 and 1.6–1.9 μM, respectively.

The K_m of the aged iron solution was also related to the total Fe concentration. The higher the concentration of iron in solution, the lower the concentration of G6P was at one-half maximum velocity. This probably accounts for the fact that we could not observe the catalytic effect in the iron solution with high concentrations (Fig. 2), and the maximum velocities of G6P hydrolysis in both 16.5 nM and 1000 nM aged Fe solutions were very similar at around 1 to 1.35 nM s⁻¹.

It is interesting to compare the catalytic behavior of aged iron solution to natural phosphoesterase and their biomimetic, though the velocities of hydrolysis G6P in aged iron solutions are much lower than that of phosphoesterase. For natural phosphoesterase, K_m and K_i of PO₄ is usually in the millimolar range;^55–60 only K_i of WO₄ and MoO₄ is in the micromolar range.^{57,58,61–63} The value of K_m of G6P is 920 μM for phosphoesterase extracted from sweet potato⁵⁰ and 300–310 μM for those from soybean seed.⁶⁴ Besides, the modes of molybdate and tungstate inhibition are noncompetitive.^{57,58,61–65} Only orthophosphate is competitive^57,58 in most cases.

A more significant difference between the aged iron solutions and the natural phosphoesterase is revealed in their response to the fluoride ion. While the activity of all known natural phosphoesterases is very sensitive to fluoride even at micromolar levels,^59,63–69 no change in the catalytic activity of these aged iron solutions were found even when the final concentration of fluoride in solutions reached 0.5 M. Another significant difference is their response to the buffer solutions. Usually, the natural phosphoesterase still has great activity within the pH range of the buffer. However, no significant promotion effect on sugar phosphate hydrolysis was observed when the buffer solution (e.g., tris, citrate) was introduced into these aged iron solutions at a similar pH (5.0–7.5) range (ESI Table 1†). The activity of these aged iron solutions decreased rapidly within 10 min, with the exception of the acetic acid-acetate buffer (the highest acetate concentration tested was 0.25 M). This is in agreement with previous observations that many buffers can significantly decrease the promotion of the metal ion on the hydrolysis of nucleoside phosphate.^29–31 All of these behaviors of the aged iron solution suggest that they are different from the natural enzyme.

It is well known that iron speciation changes due to hydrolysis during the aging process, though controversy still exists over the identity and stability of the species formed in solution, and of the interaction of iron with other species.^46,70–75 It has been suggested recently that diiron or polyiron oxide with the oxo-bridge or hydroxo-bridge (bond) might be formed in the iron solution during the aging process.⁷⁴ Based on quantum-chemical calculations using density-functional theory, dihydroxobridging binuclear compounds can be present in aqueous solutions, as binuclear dihydroxobridging [Fe(H₂O)₄(μ-OH)₂Fe(H₂O)₄]ⁿ⁺ and oxobridging [Fe(H₂O)₅(μ-O)Fe·(H₂O)₅]ⁿ⁺ (n = 2, 4) cations in the hydrolysis products of the cations [Fe(H₂O)₆]^m+ (m = 2, 3).⁷⁶ The hydroxo-bridged Fe–(OH)₂–Fe dimers are the structure units in the polymetric hydroxo complex, which are dependent on pH and aging time.^77,78 Direct experimental evidence has demonstrated the existence of dinuclear and polynuclear complexes in a 0.1 mM FeCl₃ solution. The electrospray ionization time-of-flight mass spectrometry has detected a variety of mononuclear and polynuclear iron–oxohydroxo–chloride complexes in Fe solution at a pH range of 2–6.6.⁷⁹ The (hydr)oxo-bridged Fe–Fe structure has been confirmed at the interface of iron oxide (solid) to water.^80–83 It was suggested that water reacts dissociatively on the iron oxide surface, leading to a structure with both terminal and bridge (hydr)oxy groups. The μ-(hydr)oxo ligand bridge between Fe–Fe also occurs in nanocrystalline ferrihydrite.⁸⁴ It is reasonable to assume that the aged, acid-forced hydrolysed nanomolar iron solutions comprise species with a μ-(hydr)oxo ligand bridge.

The μ-(hydr)oxo ligand bridge is a universal feature in dinuclear phosphoesterase.^10,11,15 It has been hypothesized that a “phosphoesterase motif” provides the scaffold for an active site dinuclear metal centre in the members of the phosphoesterase family.^12,85–88 They involve the cleavage of phosphoester bonds, including acid and alkaline phosphatases, bacterial exonucleases, diadenosine tetraphosphatase, 5′-nucleotidase, phosphodiesterase, sphingomyelin phosphodiesterase, which is an enzyme involved in RNA debranching, a phosphatase in the bacteriophage genome, as well as the family of ser/thr protein phosphatase (PP1, PP2A, and calcineurin).^12,15,86 Furthermore, it has also been reported that the tetrahedral oxyanions inhibit the hydrolysis of phosphate ester by these phosphoesterases.^{59,63,65–69} Moreover, the μ-(hydr)oxo ligand bridge was recognized in artificial phosphatase studies,^17–19 which yielded the principles of binucleating ligand design.^24–27 As a result, many biomimetics with capability to catalyze phosphate ester hydrolysis have been synthesized based on this special structure.^{18,21–23,25,28} It is believed that the catalytic effect of phosphatases and their biomimetics on the hydrolysis of phosphate monoesters, including sugar phosphate, is due to the net transfer of the phosphoryl group directly to water through a binuclear metal center that produces inorganic phosphate and that the nucleophile is a metal-coordinated hydroxide or the bridging hydroxo/oxo group.^{10,12,15,51,86,89} Therefore, the common feature between these aged iron solutions and the phosphoesterases (natural and synthesized biomimetics complexes) is a kind of acceleration of electron transfer rate in the structure of the μ-(hydr)oxo ligand between the metals, particularly iron.

Different nanostrutures of iron oxide have been synthesised by different laboratories,^90–95 and it has been shown that they have different functions.^96–99 One example is ferromagnetic nanoparticles with its intrinsic peroxidase activity,^96–98 with which it was demonstrated that their hydrolysis kinetics follow the Michaelis–Menten equation.^96,97 Essentially, similar to phosphoesterase, an iron center with oxo bonds is also the basic structure of many peroxidases and their mimetics.^100–106 Some PAPs were reported to have the activity of peroxidases.^107,108 Meanwhile, nanoparticles of iron oxide have been observed in the natural environment,^109–112 though some reports mentioned that the nanostructure of iron oxide can also be mediated by bacteria.^113–115 However, as recognized recently, the structure and reactivity of iron nanoparticles, even in the aqueous solution, changes with time or the aging process.¹¹⁶ Thus, it is clear that the behavior of iron oxide nanostructures in nature is very complex. It is believed that nanometre size iron oxides and (oxyhydr)oxides are ubiquitous in the earth and range from ultra-fine aerosols to precipitates or coatings in soil and sediments.^{110,117–123} Nanomolar levels of ferric ion might have existed on the ocean surface of the early earth due to photo-oxidation, even in the absence of oxygen in the atmosphere,¹²⁴ though ferrous ion (II) is presumed to be the dominant form of iron in the sea at that time.¹²⁵ The elevated CO₂ levels that are likely to have prevailed in the atmosphere at that time could have resulted in a neutral or mildly acidic ocean.¹²⁵ Moreover, it has been reported that ferric ion is present in the hydrothermal ecosystem, which is also believed to have been the realm where life first emerged.^126–130 It was also documented that magnetite (Fe₃O₄) must have been present in the Earth at that time through the process of serpentinization, i.e., the reaction of olivine- and pyroxene-rich rocks with water.^131–133

The relevance of this observation is not only to phosphorus metabolism in biology and the phosphorus cycle in geochemistry, but also to the essence of biocatalysts. It demonstrates that these aged nanomolar inorganic iron(III) solutions perform the same function as enzymes (natural and biomimetics) that have metal complexes ligated to either polypeptides or organic ligands. Natural and biomimetic phosphoesterases and aged inorganic iron solutions might have the same unique μ-(hydr)oxo bridge and metal center to perform enzymatic functions. This is supported by the concept of the metal's role as “constrained” for the selective uptake and catalytic activity in metallo-enzyme catalysis.^134–136 Pauling stated “the specificity of the physiological activity of substances is determined by the size and shape of molecules, rather than primarily by their chemical properties, and that the size and shape find expression by determining the extent to [which] these regions of the two molecules are complementary in structure... The enzyme is closely complementary in structure to the “activated complex” for the reaction catalyzed by the enzyme”.¹³⁷ Therefore, these aged nanomolar inorganic iron solutions might serve as a “primitive enzyme”¹³⁸ or as an “inorganic biocatalyst”. In other words, all of life's catalysts are enzymes, although only protein and RNA (ribozyme) are identified as enzymes in modern biochemistry.^139–144

Conclusions

Aged, acid-forced hydrolysed nanomolar inorganic iron solutions were found to have phosphoesterase activity, which significantly promoted the hydrolysis of phosphate ester following Michaelis–Menten kinetics. Moreover, the catalysis was inhibited by tetrahedral oxyanions with inhibition strength in an order of WO₄ > MoO₄ > PO₄. The activity was related to the aging process and total iron concentrations, but the detailed mechanism is still unknown. Further work is needed to understand the nature of the (hydr)oxo-bridged Fe–Fe structure in water and its potential role in organic phosphorus transformation in geochemistry. However, this observation and reported intrinsic peroxidase-like activity of ferromagnetic nanoparticles⁹⁶ demonstrates that “the chain of life is of necessity a continuous one, from the mineral at one end to the most complicated organism at the other”, as proposed by Leduc.¹⁴⁵ The hydrolysed iron solutions might be just one of ubiquitous sets of undiscovered inorganic biocatalysts (enzymes) in nature. Such inorganic enzymes might act as a bridge between the inorganic and organic worlds and would have played important roles in the origin of life. Discovery of further inorganic enzymes might provide clues on the emergence of life and a potential solution to the puzzle of “chicken and egg” in life's evolution.^{138,146–153}

Acknowledgements

X.L.H. greatly appreciates the precious comments of Drs. R.J.P. Williams, Michael J. Russell, Gerhard Schenk and Jianping Xu and personal encouragements from Drs. Tsung-Hung Peng, Peter B. Ortner, Robert Atlas and Mr. Yu-Dong Sun for this study. X. L. H also thanks Ms. Gail Derr and Dr Raghuraman Venkatapathy for English editing. Financial support for the experimental parts of the study was provided by NOAA's Center for Sponsored Coastal Ocean Research to J. Z. Z. All statements, findings, conclusions and recommendations are of the authors and do not necessarily reflect the views of Pegasus Technical Services Inc., University of Miami or NOAA or the U. S. Department of Commerce.

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Footnotes

† Electronic supplementary information (ESI) available: Fig. 1: time course of formation of phosphoantimonylmolybdenum blue complex from phosphate released during hydrolysis of 20 μM G6P in an aged-4 month, 1 μM iron solution at room temperature (22 ± 2 °C) at different hydrolysis times. Table 1: effect of tris-buffer solution on the G6P hydrolysis in an aged-14 month, 1 μM iron solution. See DOI: 10.1039/c1ra00353d

‡ Present address: Pegasus Technical Services Inc., 46 E Hollister St., Cincinnati, OH 45219, USA

§ X.-L. H. instigated this study and performed the experiments, and wrote the draft of the manuscript. X.-L. H. and J.-Z. Z. designed the experiments, and prepared the final manuscript.

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