Hydrolysis rate constant of 20 μM G6P in inorganic iron solutions.a
Phosphorus is one of the most important macronutrients for the primary production. The transformation of dissolved organic phosphorus in the environment and its contribution to biological production in the different ecosystems is still a mystery. Recently, it was demonstrated that phosphate ester can be rapidly hydrolyzed in solutions containing iron oxide nanoparticles with enzyme kinetics. The catalyst is sensitive to temperature and pH changes and inhibited by tetrahedral oxyanions with an order of PO4 < MoO4 < WO4. The oxo-Fe structure in the iron oxide nanoparticles, like the metal center of natural phosphatase (e.g., purple acid phosphatase, PAP), might contribute to the observed catalytic activity. Iron oxide nanoparticles are very common and widely exist in the current earth environment, and phosphate esters are the main component of dissolved organic phosphorus in soil and waters. It is expected that iron oxide nanoparticles in aqueous environments, as an inorganic phosphatase, play a critical role for the phosphorus transformation from the view of the phosphorus cycle.
- iron oxide
- phosphate ester
- phosphorus cycle
Phosphorus is one of the most important macronutrients for the primary production, which is primarily taken up by plants in the form of phosphate ions (HPO42− and H2PO4−). Most of the knowledge of phosphorus in the environment, including the phosphorus geochemistry cycle, comes from inorganic phosphates [1, 2, 3, 4]. The dissolved organic phosphorus transformation and its contribution to the biological production in the different ecosystems, e.g., soil, lake, estuary and ocean, is still a mystery [5, 6, 7]. Recently some limited works have indicated that different phosphate esters, especially monoesters are the main components in the dissolved organic phosphorus in soils [8, 9, 10, 11] and waters [12, 13, 14, 15, 16, 17], which might be an important source of P phytoavailability and a potential source of water eutrophication. The phosphomonoesters in supra-/macro-molecular structures were found to account for the majority (61–73%) of soil organic P in diverse agricultural soils across the world and the monoester P pool was estimated to account for 33% of the total phosphorus (587 ± 32 kg ha−1) by a recently review .
In general, the phosphate ester hydrolysis is catalyzed by various enzymes, including purple acid phosphatases (PAPs), which have been identified and characterized from plant, animal and bacterial organisms . On the other hand, several studies have already demonstrated that the phosphate ester can be hydrolyzed with the interaction of minerals in the aqueous environments [5, 20, 21, 22, 23, 24, 25]. Here, the results of laboratory study on the hydrolysis of phosphorus esters, promoted by the iron oxide nanoparticles in water, including the aged nanomolar inorganic iron ion solutions [26, 27, 28], were summarized. Additionally, the potential role of inorganic iron oxide nanoparticle for the phosphorus cycles due to the intrinsic phosphoesterase activity is postulated.
2. Promotion effect on the phosphate esters hydrolysis
Usually, phosphate ester in water is quite stable. As an example, hydrolysis Glucose 6-phosphate (G6P), a very common phosphate ester in nature, is a slow process without enzyme in the medium of deionized water (DIW), and becomes even slower in the fresh nanomolar inorganic iron solutions. Inorganic orthophosphate (Pi) in the DIW with the addition of 100 μM G6P at room temperature (22 ± 2°C) was initially 0.90 ± 0.04 μM, which became 4.86 ± 0.26 and 10.35 ± 1.19 μM at 4 and 12 days, respectively. The corresponding Pi in the fresh nanomole inorganic iron solutions (0.5–50 nM Fe(NO3)3) were 1.35 ± 0.09 and 2.55 ± 0.15 μM.
After G6P was added into an aged 14-month 16.5 nM Fe(NO3)3 solution (pH 6.30) at room temperature, made by acid-forced hydrolysis , the Pi was rapidly released (e.g. the initial 20 μM G6P, as presented in Figure 1). Like metal ions as well as natural and biomimetic enzymes, the kinetics of G6P hydrolysis in the aged iron solution can be described as a pseudo-first-order reaction for a fixed concentration of G6P [29, 30, 31, 32, 33, 34, 35, 36]. For the initial 20 μM G6P, the decrease in G6P concentration, [G6P]
The corresponding reaction rate constant (
Like aged inorganic iron solution, the concentration of phosphate esters and condensed inorganic phosphate decreased, and inorganic orthophosphate (Pi) increased in a solution bearing iron oxide (IO) nanoparticles, which consists of a dialysis membrane tube (DMT, e.g., Spectra/Por 1 membranes, molecular weight cut-off (MWCO) 6000–8000 Da) filled with iron oxide (DMT-IO). The iron oxide (D) was synthesized by Fe(NO3)3 following the basic protocol of Atkinson  and aged at 80°C . The
|Fe source||Manufacturer||Aged time (mo.)||Aged temperature (°C)||IO nanoparticles or total Fe concentration (nM)||20 μM G6P|
|Iron oxide nanoparticles (IO)||Fe(NO3)3||JT Baker||0.25||5||A||16.9||11|
|Aged acidic forced hydrolysis inorganic Fe solution||Fe(NO3)3||JT Baker||14||22||16.5||30.17||6.4|
|Iron standard solution (metal Fe in 0.3 M HNO3)||JT Baker||4||22||1||2.38||81.0|
These inorganic iron solutions also have the same promotion effects on hydrolysis of different sugar phosphates, including G2P, ribose-5-phosphate (5-carbon, R5P), and fructose 1-phosphate (6-carbon, F1P) (Table 2). As expected, the promotion effect was also found on the hydrolysis of AMP, ADP and ATP, and inorganic condense phosphates (poly-Pi and PPi) as well as the RNA model compound (4-nitrophenyl phosphate ester, pNPP). 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).
|Phosphate ester||Fe source||Initial OP (μM)||Rate constant ||Half-life |
|Glycerol-2-phosphate (G2P)||Fe standard solution, 7.5 nM, 4 mo.||10||12.69||15.2|
|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|
|IO-D (made by Fe(NO3)3, aged a week at 80°C, soak 1 month at 22°C)||10||85.02||2.26|
|Ribose-5-phosphate (R5P)||Fe standard solution, 7.5 nM, 4 mo.||10||13.92||13.8|
|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|
|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||8.5||22.6|
|Adenosine monophosphate (AMP)||IO-D (made by Fe(NO3)3, aged a week at 80°C, soak 1 month at 22°C)||10||79||2.44|
|Adenosine diphosphate (ADP)||IO-D (made by Fe(NO3)3, aged a week at 80°C, soak 1 month at 22°C)||10||134||1.44|
|Adenosine triphosphate (ATP)||IO-D (made by Fe(NO3)3, aged a week at 80°C, soak 1 month at 22°C)||10||61.5||3.13|
|IO-A(made by Fe(NO3)3, aged a week at 5°C, soak 1 month at 22°C)||20||19.6||9.8|
|IO-B (made by Fe(NO3)3, aged a week at 22°C, soak 1 month at 22°C)||20||40.4||4.8|
|IO-G (made by FeCl3, aged a week at 80°C, soak 1 month at 22°C)||20||38.7||5.0|
|Polyphosphate (poly-Pi)||IO-D (made by Fe(NO3)3, aged a week at 80°C, soak 1 month at 22°C)||10||67.5||2.85|
|Pyrophosphate (PPi)||IO-D (made by Fe(NO3)3, aged a week at 80°C, soak 1 month at 22°C)||10||162||1.19|
As expected, the catalytic activity is related to the soaked time of DIW with DMT-IO and the nature of IO, which can be described by the hydrolysis reaction rate constant. The kinetics of
3. Kinetics of hydrolysis phosphate esters
As presented in Table 2, the hydrolysis reaction rate constant at different initial concentrations of phosphate esters in these aged inorganic iron salt solutions or inorganic iron oxides solutions were not constant. Surprisingly, the
In fact, the promotion effect of G6P hydrolysis can be extended to 2500 μM in this aged iron solution with a
The same patterns were also observed in the solution bearing inorganic iron oxide nanoparticles. Like the aged inorganic iron solution, the
|Phosphorus source||Range (μM)|
It should further be pointed out that the similar enzyme kinetics (Michaelis-Menten equations) were observed recently by many inorganic nanoparticles studies, which have been described as nanozyme [44, 45, 46, 47]. For example, Fe3O4 , α-Fe2O3 , γ-Fe2O3 , γ-FeOOH , Co3O4 , MnFe2O4 [52, 53], MFe2O4 (M = Mg, Ni, Cu) , ZnFe2O4 , NiO , and MnO2  have been observed to have peroxidase-like or catalase-like activity, whereas the vanadium pentoxide (V2O5) was demonstrated to have antioxidant enzyme-like (glutathione peroxidase) activity [58, 59, 60, 61] and molybdenum trioxide (MoO3) nanoparticles to have sulfite oxidase activity .
4. Inhabitation effects of tetrahedral oxyanions
The hydrolysis of phosphorus ester was significantly inhibited when the tetrahedral oxyanions were introduced into inorganic iron oxides nanoparticle solution, e.g., G6P in a 10-month aged iron solution (Figure 4), as the natural PAPs. Both the catalytic and the inhibition behaviors of the catalysis in the presence of 5–125 μM G6P with different tetrahedral oxyanions can be described by a Michaelis-Menten equation (Eqs. (3)–(7)) as follows:
The results indicated that the catalysis sites from these catalyst, i.e., the inorganic iron oxide nanoparticles, may be only bound to either the tetrahedral oxyanions (PO4, MoO4, and WO4) or phosphate esters to form an intermediate, but cannot bind both of them at any given moment. The modes of tetrahedral oxyanions and G6P are competitive (Figure 5). The
A more significant difference between the inorganic catalyst and the natural phosphoesterase is revealed in their response to the fluoride ion. The activity of all known natural phosphoesterase is very sensitive to fluoride, even at the micromolar level [67, 72, 73, 74, 75, 76, 77, 78], while the catalytic activity of the inorganic iron oxides solutions still remain, even when the final concentration of fluoride in the solutions were up to 0.5 M.
5. Effect of temperature
The catalyst on the hydrolysis of phosphate ester is sensitive to temperature, as natural enzymes. The optimum temperature for the phosphate ester hydrolysis reaction by these inorganic catalysts was around 50°C (Figure 6), which is comparable to recent observations on the natural enzymes [79, 80, 81, 82]. However, catalytic activity of the IO nanoparticles in solution was lost as the temperature was raised to 90°C for an hour or to 72°C for 16 h. This behavior is similar to the thermal denaturation of the natural enzyme. Moreover, the temperature coefficient,
In actuality, the catalytic activity of the nanoparticles remained high after removal from their source (IO) for days, even when stored at −18°C, demonstrated by a storage experiment (Table 4) , which further suggested that IO nanoparticles can be displaced to a considerable distance from their source and still maintain catalytic activities for a considerable time. Meanwhile, low temperatures, even frozen conditions, also favor the persistence of catalytic activity from these IO nanoparticles. These are important from the view of astrobiology (origin of life) , but also for plant acquisition, nanoengineering and the potential application for industrial production.
|Treatment||20 μM G6P||20 μM ATP|
|1 h Pi (μM)||5 h Pi (μM)||1 h Pi (μM)||5 h Pi (μM)|
|11 days at 22°C||11R||5.49 ± 0.004||13.89 ± 0.064||3.2||4.47 ± 0.016||9.87 ± 0.255||6.0|
|11 days at 4°C||11 L||5.51 ± 0.010||13.64 ± 0.042||3.3||4.36 ± 0.005||9.71 ± 0.066||6.3|
|5 days frozen (−18°C) and 6 days at 4°C||5F6L||4.57 ± 0.013||11.48 ± 0.030||4.5||3.76 ± 0.009||7.61 ± 0.043||9.4|
|5 days at 4°C and 6 days at 22°C||5L6R||5.85 ± 0.011||14.10 ± 0.211||3.1||4.51 ± 0.018||10.30 ± 0.123||5.7|
|5 days at 50°C and 6 days at 22°C||5H6R||2.93 ± 0.01||6.68 ± 0.020||11||2.71 ± 0.012||4.92 ± 0.032||19|
|5 days at 50°C and 6 days at 4°C||5H6L||2.62 ± 0.002||5.44 ± 0.001||16||2.48 ± 0.002||3.72 ± 0.04||36|
|9 days at 50°C and 2 days at 22°C||9H2R||2.43 ± 0.005||4.07 ± 0.005||26||2.38 ± 0.016||3.08 ± 0.025||60|
6. Effect of pH and buffer solution
pH is another key factor for enzyme activity. The aged inorganic iron solution or the water bearing iron oxide nanoparticles, e.g., DMT-IO, are generally mildly acidic (pH 5.5–6.5). Various concentrations of bicarbonate were introduced in the DMT-IO system, but in all cases enzyme-like activity for phosphate ester hydrolysis remained quite high (Figure 7a). In general, the most favorable pH of the enzyme-like activity was found to be between 6 and 7, though the phosphorus source, the concentrations of bicarbonate, and the type of DMT-IO also influenced its activity (Figure 7b and c). When pH was raised beyond 7 (e.g., pH 7, 7.2 and 8), the catalysis coefficient,
It should be pointed out that the catalysis capacity of these solutions bearing inorganic iron oxide nanoparticles is closely related to the buffer used in the system. The catalytic activity dropped precipitously after a small amount of citrate buffer (pH 4.0–6.2) or tris(hydroxymethyl)-aminomethane (TRIS) (pH 5.8–7.2) was introduced into an inorganic iron solution (Table 5). Both citrate [83, 84] and TRIS  can react with Fe (III) in solution to form the aqueous Fe-complex, particularly at high ratios of citrate or TRIS to Fe (>>10,000:1 molar ratio). But the catalysis of these solutions does not change when the acetic acid-acetate buffer system was introduced, the final acetate concentration in the solution was up to 0.25 M. All of these responses imply that the significant change was not due to the pH itself, but to the interactions between nanoparticles in the solution and chemicals in the environment. It has been reported that some buffer systems can significantly inhibit the activity of natural enzymes, for example, citrate on special PAPs  and alkaline phosphatase  and TRIS on aminopeptidase and RimO methylthiotransferase [88, 89] due to structure changes and metal-complex formation [84, 85, 90].
|Treatmenta||Initial G6P (μM)||Reaction time (h)||Pi (μM)||Rate constant ||Half-life |
|DIW and aged 14 mo., 1000 nM, Fe(NO3)3, (1:1)||20||0||0.22||22.1||8.7|
|Aged 14 mo., Fe(NO3)3, 1000 nM and tris–HCl buffer (10 mM, pH 7.0), 1:1||20||0||0.22||1.72||111.8|
|DIW and aged 14 mo., 1000 nM, Fe(NO3)3, (1:1)||50||0||0.44||11.3||17.1|
|Aged 14 mo., Fe(NO3)3, 1000 nM and tris–HCl buffer (10 mM, pH 7.0), 1:1||50||0||0.44||1.93||99.8|
7. Natural and inorganic phosphatase
Recall the natural phosphatase, a binuclear metal center (di-iron Fe-Fe or Fe-M (M as Mn and Zn)) that produces orthophosphate due to the net transfer of the phosphoryl group to water, is essential for its catalysis (Figure 8a) [19, 66, 91, 92, 93, 94]. The μ-(hydr)oxo ligand bridges in the metal center—the key of “phosphoesterase motif”—are a universal feature in binuclear phosphoesterase [19, 94]. They are responsible for the cleavage of phosphoester bonds; including acid and alkaline phosphatases; bacterial exonucleases; diadenosine tetraphosphatase; 5′-nucleotidase; phosphodiesterase; sphingomyelin phosphodiesterase, an enzyme involved in RNA debranching; and a phosphatase in the bacteriophage genome as well as for the family of Ser/Thr protein phosphatase (PP1, PP2A, and calcineurin) [95, 96]. Based on the μ-(hydr)oxo metal bridge structure, different artificial phosphatases have been synthesized by using different organic ligands to stabilize the metal center [19, 97, 98].
It is well known that iron speciation changes due to ion (III) hydrolysis in the solution during the aging process, diiron or polyirons oxide with the oxo-bridge or hydroxo-bridge (bond) might be formed [99, 100, 101, 102, 103]. Based on the quantum-chemical calculations by 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 cations [Fe(H2O)6]m+ (m = 2, 3) . 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 [105, 106]. Molecular dynamics simulation further demonstrated the presence of aqueous di-iron or poly-irons, in which the Fe-Fe distance is 3.0–3.5 Å, with bonds by oxo-bridge or hydroxo-bridge . Meanwhile, the (hydr)oxo-bridged Fe-Fe structure has been confirmed by experiments in the interface of iron oxide (IO, solid) to water [108, 109, 110, 111]. The μ-oxo iron ion have been identified in-situ in the high concentrated inorganic iron solution (e.g., 0.1 M Fe(NO3)3 , and 0.1 M FeCl3 . The solubility of IOs further indicates that the critical ferrihydrite nucleus with an equivalent diameter of ∼15 Å and containing only ∼30 Fe atoms is stable in aqueous solution . The 10-angstrom discrete iron-oxo cluster (known as the Keggin ion, Fe13) is also soluble , as a constitute structure of ferrihydrite nanoparticles . Consequently, iron oxide nanoparticles with 3.5 Å oxo-Fe bindings (e.g., doubly shared iron octahedra) such as ferrihydrite, goethite, hematite, magnetite, and even green rust (fougerite) can be presented in the natural environment (Figure 8b) [115, 116, 117, 118, 119, 120, 121]. Therefore, it is reasonable to suggest that the oxo bridged Fe-Fe structure in the aqueous IO nanoparticles contribute to the catalysis of phosphate ester hydrolysis . This compares to the activity of the aged nanomolar inorganic iron ion solutions [26, 27] and mimics of the artificial phosphatases [19, 97, 98]. The common feature between these IO nanoparticles, either from the DMT-IO or the aged inorganic iron ion solutions, as well as the natural or synthesized biomimetic phosphoesterase, constitute a kind of acceleration of electron transfer rate in the structure of the μ-(hydr)oxo ligand between the metals, particularly iron [19, 26, 27, 28, 92, 94, 122, 123]. In other words, the hydrolysis of phosphate ester is entirely dependent on its catalysis on this special Fe-oxo-Fe structure [27, 28]. Experiments and chemical models have also demonstrated that temperature impacts the stability of the aqueous poly-iron formation  and the nanostructure of IO in the solution [117, 125], which can explain the thermal denaturation behavior of the inorganic phosphatase (Figure 6). The Fe-Fe structure in the nanoparticles due to the nanosize-induced phase transformation and changes in the IO nanoparticle solution with the dissolved CO2  further supported the response of the inorganic phosphatase at different pH (Figure 7).
Similar to phosphatases, the active metal centers of most peroxidase and catalases in nature also comprise the transition metals, for example, horseradish peroxidase, HRP , heme catalases , uroerythrin  with Fe, manganese peroxidase , manganese catalases [131, 132] with Mn or haloperoxidases  with V, all exhibit the oxo ligand structure. This unique structure might be also accountable for the “intrinsic peroxidase or catalases” from different inorganic metal oxides nanoparticles [44, 48, 49, 51, 53, 54, 58, 59, 60, 61, 134, 135]. It was noted that some PAPs were also reported to have activity of peroxidases [136, 137]. The
Several recently studies from Europe have suggested that iron-rich nanoparticles (<20 nm) are the main carriers of phosphorus in forest streams and soil solution [10, 11, 144, 145] and monoesters are the main composition of dissolved organic phosphorus in soil and water [10, 15, 16, 18]. This further imply that iron oxide nanoparticles might play a significantly role for the organic phosphorus transformation from the view of phosphorus biogeochemistry, although sorption and precipitation is still the dominant view of the current soil and environmental science on the interaction of iron oxides and dissolved organic phosphorus in soil and sediment [21, 146, 147, 148, 149]. A couple of studies still noticed that orthophosphate can be released during the processing of the interactions [5, 20, 21, 22, 23, 24, 25]. As iron oxide nanoparticles are very common and widely exist in the soil, sediment, dust, and water [125, 150, 151, 152, 153], such enzyme-like catalytic propensities on phosphate esters in the current earth environment may provide an undiscovered feedback of organic phosphorus and play a critical role in the phosphorus cycles.
On the other hand, many effects have been made to improve inorganic nanozyme, both its catalysis capacity and substrate specificity, particularly for the “engineering peroxidase” related to iron oxide for its analytical, biomedical, and environmental applications from the view of nanoengineering [46, 47, 154, 155]. Various polymers or other organic compounds, e.g., porphyrin rings, the backbones of short peptides, amino acids, and even DNA, have been employed in the stabilization of the oxo bridged Fe-metal center in different iron oxides [156, 157, 158, 159, 160]. Similar effects should be made for the inorganic phosphatase as well. These “engineering phosphatase” can be employed for environmental monitors after standardization to assess the availability of dissolved organic phosphorus in waters and its potential risk for water eutrophication due to its higher stability and lower cost than protein enzymes, supported by the fact that natural phosphatase has been used for the tool to assess water or soil phosphorus availability [161, 162, 163, 164]. Another possibility for industry is to use these high efficiencies engineered phosphatase to release the orthophosphate from the wastewater directly for agriculture.
8. Conclusions and future prospective
Laboratory experiments on the hydrolysis of phosphate ester in water demonstrated that inorganic phosphoesterase-like activity, using various inorganic iron oxide nanoparticles, significantly promotes the hydrolysis of phosphate ester, including G6P, PPi, and ATP. These findings and the fact that this and other inorganic nanoparticles can act effectively as enzymes: for example, iron oxide as peroxidase, vanadium pentoxide as bromoperoxidase, and molybdenum trioxide nanoparticles as sulfite oxidase; further support the concept of inorganic enzymes. The catalytic property of these nanoparticles is likely due to the structure of the metal oxides or metal bonds in the oxides and not merely to the nanoparticle surfaces. As iron oxide nanoparticles are very common and widely exist in the soil, sediment, and water, such enzyme-like catalytic propensities on phosphate esters, the main composition of dissolved organic phosphorus, in the current earth environment may play a critical role in the phosphorus cycles.
This work is dedicated to my beloved parents (Dr. Jing-Xiong Ji and late Dr. Shi-Xiong Huang) and my family (Wei Sun and Jack Jixiang Huang) for their love, endless support, encouragement & sacrifices. X.L.H. greatly appreciates the precious comments from the late Dr. R.J.P. Williams in the past years, and the kindly permissions to use the structure of iron oxide (Figure 8b) from Dr. Jean Pierre Jolivet as well as the personal encouragements from Drs. Robert Atlas, Gerhard Schenk, Michael J. Russell, Jia-Zhong Zhang, Raghuraman Venkatapathy and Peter B. Ortner. The experiment part of this work was initially conducted by X.L H from 2007 to 2008 at the AOML, NOAA, supported by the National Oceanic and Atmospheric Administration’s (NOAA) Coastal Ocean Program and Climate and Global Change Program. The research was carried out, in part, under the auspices of the Cooperative Institute of Marine and Atmospheric Studies (CIMAS), a joint institute of the University of Miami and NOAA, cooperative agreement #NA67RJ0149. The statements, findings, conclusions, and recommendations are those of the author and do not necessarily reflect the views of CIMAS, NOAA or the U.S. Department of Commerce.
Conflict of interest
The author declares no competing financial interest.