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, 91–94]. 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

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

#### Figure 8.

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

Interaction between Tris-HCl buffer and aged inorganic iron solution on the hydrolysis rate of G6P.a

Buffer experiments were conducted with a 1000 nM aged (14-month) inorganic iron solution. The iron solution diluted by DIW (1:1) was used as a control. Either 4 ml of 10 mM Tris-HCl (pH 7.0) or 4 ml of DIW was mixed with a 4 ml of aged iron solution, then 0.1 ml of 1.6 or 4.0 mM G6P stock solution was added to make a final solution containing 20 or 50 μM G6P. Pi concentration was determined at 0, 0.17, 1, 2 and 6 h, respectively, after the addition of G6P. The results of Pi in the table was an average of three replications. The rate constant and half-life were

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] due to structure changes and metal-complex forma-

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

tion [84, 85, 90].

108

7. Natural and inorganic phosphatase

Treatment<sup>a</sup> Initial

DIW and aged 14 mo., 1000 nM,

Aged 14 mo., Fe(NO3)3, 1000 nM and tris–HCl buffer (10 mM, pH 7.0), 1:1

DIW and aged 14 mo., 1000 nM,

Aged 14 mo., Fe(NO3)3, 1000 nM and tris–HCl buffer (10 mM, pH 7.0), 1:1

calculated based on the first order kinetics.

Fe(NO3)3, (1:1)

Nanocatalysts

Fe(NO3)3, (1:1)

a

Table 5.

G6P (μM) Reaction time (h)

Pi (μM)

20 0 0.22 22.1 8.7

20 0 0.22 1.72 111.8

50 0 0.44 11.3 17.1

50 0 0.44 1.93 99.8

1 1.75 2 3.37 6 7.74

1 0.35 2 0.41 6 0.93

1 2.45 2 4.58 6 11.14

1 0.71 2 0.99 6 2.45

Rate constant k (10<sup>6</sup> s 1 )

Half-life t0.5 (h)

The metal center of phosphatase. (a) The μ-(hydr)oxo-bridges in purple acid phosphatase (PAP), and (b) Fe-Fe structure in different iron oxides phases.

phosphatases; bacterial exonucleases; diadenosine tetraphosphatase; 5<sup>0</sup> -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 [99–103]. 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) [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 [108–111]. 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 [101], and 0.1 M FeCl3 [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, Fe13) 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) [115–121]. 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 CO2 [126] further supported the response of the inorganic phosphatase at different pH (Figure 7).

On the other hand, many effects have been made to improve inorganic nanozyme, both its catalysis capacity and substrate specificity, particularly

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

wastewater directly for agriculture.

Acknowledgements

U.S. Department of Commerce.

111

8. Conclusions and future prospective

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

ronment may play a critical role in the phosphorus cycles.

for the "engineering peroxidase" related to iron oxide for its analytical, biomedical,

employed in the stabilization of the oxo bridged Fe-metal center in different iron oxides [156–160]. 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 [161–164]. Another possibility for industry is to use these high efficiencies engineered phosphatase to release the orthophosphate from the

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 envi-

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

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 Km 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 H2O2 in the environment, or they have a much higher affinity for phosphate esters or H2O2 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 H2O2 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 V2O5 nanomaterials during H2O2 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, 146–149]. A couple of studies still noticed that orthophosphate can be released during the processing of the interactions [5, 20–25]. As iron oxide nanoparticles are very common and widely exist in the soil, sediment, dust, and water [125, 150–153], 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.

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

[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 nano-

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 [126] further supported the

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 Km 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 H2O2 in the environment, or they have a much higher affinity for phosphate esters or H2O2 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 H2O2 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 V2O5 nanomaterials during H2O2 catalysis cycle further demonstrated that the catalytic characteristics in these nanoparticles is directly related to the metal structure in the nanoparticle surface

structure of IO in the solution [117, 125], which can explain the thermal

response of the inorganic phosphatase at different pH (Figure 7).

Nanocatalysts

[61], which supports the concept of inorganic enzyme [27, 28].

play a critical role in the phosphorus cycles.

110

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, 146–149]. A couple of studies still noticed that orthophosphate can be released during the processing of the interactions [5, 20–25]. As iron oxide nanoparticles are very common and widely exist in the soil, sediment, dust, and water [125, 150–153], such enzyme-like catalytic propensities on phosphate esters in the current earth environment may provide an undiscovered feedback of organic phosphorus and

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 [156–160]. 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 [161–164]. 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.

#### Acknowledgements

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.

Nanocatalysts
