Iron Oxide Nanoparticles: An Inorganic Phosphatase

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 PO 4 < MoO 4 < WO 4 . 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.


Introduction
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 (HPO 4 2À and H 2 PO 4 À ). 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 AE 32 kg ha À1 ) by a recently review [18].
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 [19]. 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.

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 (P i ) in the DIW with the addition of 100 μM G6P at room temperature (22 AE 2°C) was initially 0. 90  After G6P was added into an aged 14-month 16.5 nM Fe(NO 3 ) 3 solution (pH 6.30) at room temperature, made by acid-forced hydrolysis [27], the P i 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] t , due to its hydrolysis can be expressed as a function of hydrolysis time, t, as log G6P ½ t ¼ À1:31 Â 10 À5 t À 4:718 r 2 ¼ 0:999 À Á (1) where [G6P] t is in M and t is in second. The corresponding reaction rate constant (k) was 3.02 Â 10 À6 s À1 , and the half-life (t 1/2 ) was 6.38 h. Similar to the initial 20 μM of G6P, the P i concentration of a initial 100 μM of G6P in the aged inorganic iron solution at 1, 3 and 6.7 h was 4.95, 10.74 and 20.62 μM, respectively. The corresponding k was 8.83 Â 10 À6 s À1 , and the t 1/2 was 21.8 h. It is highlighted that these k in the aged iron solution were much higher than the previously reported rates in the presence of the fresh unaged nanomolar inorganic iron [26] and millimole metals [30-32] solutions.
Measured k of the initial 20 μM G6P with different sources of iron, either the aged inorganic iron solutions or the solutions bearing inorganic iron oxide nanoparticles (DMT-IO), are listed in Table 1. The half-life for aged 4-month Fe (NO 3 ) 3 (16.5 nM), FeCl 3 (10 nM) and Fe(ClO 4 ) 3 (10 nM) was 37.8, 58.6 and 78.4 h, respectively, whereas the half-life of IO from the same source (Fe(NO 3 ) 3 , JT Baker), aged at 5-80°C was 11, 2.7, 3.2 and 2.8 h, though they were in the same order of magnitude. The same patterns were observed for the ATP as well [28]. These results further indicate, as expected, that the behavior of catalysis depends on the sources of iron oxides nanoparticles in solutions-whether FeCl 3 , or Fe(NO 3 ) 3 , and even on the different manufacturers, as well as with the different aging temperatures for IO (5-80°C) [37,38]. No clear relationships between ferric ion (III) sources, age processing, and catalytic activity, with the hydrolysis rate constant, were observed.
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-P i and PP i ) 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).
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 k of 100 μM G6P and ATP in three different IOs is presented in Figure 2. These results can be explained by the changes of the nanoparticles concentration in the water. It was expected that the concentration of the IO nanoparticles in these solutions would initially increase up to 10 days and then reach equilibrium. However, the total dissolved iron concentrations in these solutions were still beneath the detection limits of iron (0.1 nM) [39].

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 k from 5 to 250 μM G6P in the 16. 5   1 In fact, the promotion effect of G6P hydrolysis can be extended to 2500 μM in this aged iron solution with a k of 6.53 Â 10 À7 s À1 , and t 1/2 of 295 h. It should be pointed out that the concentration of total phosphorus in the solution was 10 3 -10 5 higher than that of iron in the solution (e.g., 16.5 nM Fe and 2500 μM G6P).
The same patterns were also observed in the solution bearing inorganic iron oxide nanoparticles. Like the aged inorganic iron solution, the k of various organic phosphate esters or condensed phosphates at different concentrations were not  Table 1). constant ( Table 2). The catalytic activity of the different concentration of phosphorus also can be described by the typical Michaelis-Menten equations ( Figure 3c). Based on the Lineweaver-Burk linear equation (1/V is a linear function of 1/[S]), the Michaelis-Menten constant (K m ) and maximum velocity (V m ), as well as the range of concentration of phosphorous among these compounds, were determined (Table 3). Meanwhile, the catalysis activity was still observed even when the total phosphorus esters exceeded the range of the Michaelis-Menten equations, as with many of the natural enzymes, including the PAP [43].
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]

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: without any addition : with 5 μM PO 4 : with 10 μM PO 4 : 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 (PO 4 , MoO 4 , and WO 4 ) 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 K m , the G6P concentration at which the reaction rate reaches one-half of maximum velocity (v max /2), was about 2.7 μM G6P in this aged iron solutions with no inhibitors. The K Mapp with addition 1 μM WO 4 , MoO 4 , and 5 and 10 μM PO 4 was 46.2, 14.1, 11.1 and 17.1 μM G6P, respectively. Therefore, the K i of WO 4 , MoO 4 , and PO 4 are 0.06, 0.24 and 1.6-1.9 μM, respectively. It is interesting to compare the catalytic behavior of these inorganic iron oxides nanoparticles solution to natural PAPs and their biomimetics, though the velocities of hydrolysis G6P in the inorganic catalyst are still lower than that of natural phosphoesterase. For natural PAPs, K m and K i of PO 4     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.

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, Q 10 , a measure of the hydrolysis velocity, is also decreased as a consequence of increasing the temperature by 10°C. This effect too, is comparable to the general patterns of enzyme behavior in biological systems [80,81]. Taken together, these observations also carry the implication that moderate, i.e., 50°C, and not high temperatures, were likely favorable to the catalytic reactions from the view of efficiency and speed of the catalyst.
In actuality, the catalytic activity of the nanoparticles remained high after removal from their source (IO) for days, even when stored at À18°C, demonstrated by a storage experiment ( Table 4) [29], which further suggested that IO nanoparticles can be displaced to a considerable distance from their source and still  Table 1). 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) [28], but also for plant acquisition, nanoengineering and the potential application for industrial production.

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 enzymelike 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, k, decreased as the concentration of HCO 3 increased, especially for the DMT-IO-D. When pH in solution was <7 (e.g., pH 6.2, 6.4, and 6.8), however, there were no clear patterns of k with respect to the concentration of HCO 3 and both the ATP and G6P in these two nanoparticles-bearing solutions. At the same time, k at weak acidic conditions (pH 6.2-6.8) was much higher than at weak base conditions (pH 7-8). This conclusion was further supported by an additional experiment involving the hydrolysis of ATP, whereby the pH values of DMT-IO solution were extended from 4 to 9.   Table 4.

Effect of storage conditions on the catalysis activity of iron oxide (IO-F) nanoparticle solutions. a
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 1).
TRIS [85] 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 [86] and alkaline phosphatase [87] and TRIS on aminopeptidase and RimO methylthiotransferase [88,89]

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, 91-94]. 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   Table 5.
Interaction between Tris-HCl buffer and aged inorganic iron solution on the hydrolysis rate of G6P. a phosphatases; bacterial exonucleases; diadenosine tetraphosphatase; 5 0 -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] 6 ] m+ (m = 2, 3) [104]. 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 [107]. 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(NO 3 ) 3 [101], and 0.1 M FeCl 3 [112]. 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 [113]. The 10-angstrom discrete iron-oxo cluster (known as the Keggin ion, Fe 13 ) is also soluble [114], as a constitute structure of ferrihydrite nanoparticles [102]. 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 [28]. 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, 26-28, 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 [124] 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 CO 2 [126] 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 [127], heme catalases [128], uroerythrin [129] with Fe, manganese peroxidase [130], manganese catalases [131,132] with Mn or haloperoxidases [133] 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, 58-61, 134, 135]. It was noted that some PAPs were also reported to have activity of peroxidases [136,137]. The K m in Table 3, which denotes the affinities of the phosphate ester to catalysis, are significantly (up to three orders of magnitude) lower than that of natural PAPs [33, 66, 69, 91, 138, 139]. The same patterns were also observed for these inorganic peroxidases compared to its corresponding HRP [28]. This finding further indicates that the IO nanoparticles are either much more sensitive to the low concentration of phosphate ester or H 2 O 2 in the environment, or they have a much higher affinity for phosphate esters or H 2 O 2 compared to the natural enzymes, although the maximum velocity of the hydrolysis was relatively low with these IO nanoparticles, especially for high phosphate ester or H 2 O 2 concentrations in the environment. Like the nanoparticles of IO and vanadium pentoxide, the intrinsic sulfite oxidase activity of molybdenum trioxide nanoparticles is also due to the oxo ligand of Mo, as revealed in the metal center of sulfite oxidase [62, 140,141]. Essentially, the catalytic activities depend to some degree on the surface area of these nanoparticles, but not just merely on particle size [48,51,52,134,142,143]. The in situ Raman spectroscopy on the changes of V-oxo (V=O) bond in the different V 2 O 5 nanomaterials during H 2 O 2 catalysis cycle further demonstrated that the catalytic characteristics in these nanoparticles is directly related to the metal structure in the nanoparticle surface [61], which supports the concept of inorganic enzyme [27,28].
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.

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, PP i , 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.