Nano-sized manganese oxide–bovine serum albumin was synthesized and characterized. It is promising and biomimetic catalyst for water oxidation

Abstract

Nano-sized manganese oxide–bovine serum albumin was synthesized and characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), UV-vis, X-ray diffraction (XRD), infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), elemental mapping and transmission electron microscopy (TEM). The oxide shows water oxidizing activity in the presence of cerium(IV) ammonium nitrate as a non-oxo transfer oxidant. We used bovine serum albumin in the compound as a promising model for stabilizing proteins in photosystem II.

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Replacing fossil fuels by an energy system that uses sustainable fuel is among the most critical issues of today's society.¹ Water splitting into H₂ and O₂ is currently much discussed as a promising route for the conversion of solar, wind, ocean current, tidal or wave energy into hydrogen as a “solar fuel”.² One of the great challenges to make such a plan is the development of efficient catalysts for the oxidation of water to molecular oxygen.^2–4

Plants, algae and cyanobacteria use light energy to oxidize water efficiently.⁵ Water oxidation is catalysed by a Mn₄CaO₅ cluster known as a water oxidizing complex (WOC), or an oxygen-evolving complex (OEC), housed in photosystem II (PSII).^6–8

Since the WOC consists of four manganese ions,^7,8 particular attention has been given to the manganese compounds aimed at simulating the WOC of PSII.⁹ Few manganese complexes have been discovered so far that can act as homogeneous catalysts for the oxidation of water.⁹ However, manganese oxides have been reported as heterogeneous catalysts for water oxidation by Shilov and Shafirovich, Morita et al., Harriman et al. and other groups.¹⁰ Recently, in a different approach aimed at simulating the Mn₄CaO₅ cluster in PSII, we introduced layered manganese oxides as the closest structural and functional analogs to the WOC in PSII found so far.¹¹ However, these synthetic models contain no amino acid groups to stabilize manganese oxide, proton transfer and/or decrease the activation energy for water oxidation as in PSII.^7,8,12

There are at least six intrinsic proteins that appear to be required for water oxidation by the WOC in PSII. These are proteins CP 47, CP 43, the D1 protein, the D2 protein, and the α and β subunits of cytochrome b₅₅₉.¹² Thus, PSII consists of hundreds of amino acids (Fig. 1), but only a small fraction of the residues come in to direct contact with the manganese–calcium cluster (Fig. 1).¹²

Fig. 1 The Mn₄CaO₅ cluster and the surrounding amino acids. The whole structure of the Mn₄CaO₅ cluster resembles a distorted chair, with the asymmetric cubane. There is only a small fraction of the residues that come in to direct contact with the manganese–calcium cluster (top). A three dimensional image of the bovine serum albumin molecule. The imidazole groups as proposed sites for the interaction with manganese ions are shown in yellow (bottom).

Many of the amino acids in PSII are involved in proton, water or oxygen transfer. The roles for the residues that come in contact directly with the manganese–calcium cluster include the regulation of charges and the electrochemistry of the Mn–Ca cluster, and helping coordinate water molecules at the appropriate metal sites and/or the stability of this cluster.¹²

The PSII manganese stabilizing protein is a highly conserved extrinsic component of the WOC and its deletion from the photosystem could cause a dramatic lowering of the rate of oxygen evolution.¹² The PSII manganese stabilizing protein in addition to stabilizing manganese oxides and/or decreasing the activation energy for water oxidation, has also been suggested to be important in linking the active site of the WOC with the lumen and to be involved in the proton transfer network.¹² Here, for the first time a nano-sized manganese oxide–bovine serum albumin (BSA) is studied as a structural and functional model for the WOC in PSII. BSA was used in the compound as a promising model for stabilizing proteins in PSII.

BSA is one of the most studied proteins that has a strong affinity for a variety of inorganic molecules binding to different sites.¹³ BSA is a soluble protein in the plasma of the circulatory system and acts in the transport and deposition of endogenous and exogenous substances. Albumin can readily undergo conformational changes, and is classed as a soft protein due to its relatively flexible structure. The three dimensional image of the serum albumin molecule is shown in Fig. 1.¹⁴

The charge, coulombic forces, van der Waals forces, hydrophobic interactions, sorbate conformational stability and surface area that the particle provides are important factors for the interaction of a protein and a compound.¹⁵

The nano-sized manganese oxide–BSA, reported here, was synthesized by a very simple method. In brief, manganese(II) chloride (80.0 mg, 0.62 mmol) was added to a solution of BSA (0.5 g) in water (40.0 mL) and the solution was stirred for four hours. Then to the solution, KMnO₄ (44 mg in 8.0 mL water) was added under stirring. After evaporating water, the manganese oxide–BSA film was obtained. Nano-sized manganese oxide–BSA as both a solution and film shows water oxidizing activity.

The brown solution was characterized by UV-vis, SEM, TEM, IR, AFM, and XRD. The water oxidation activity of the compound was studied. Drying the solution in air and at room temperature produced a homogenous nano-sized manganese oxide–bovine serum albumin film (see the ESI†). In the IR spectrum of the compound, the broad band at ∼3200–3500 cm⁻¹ related to the antisymmetric and symmetric O–H stretchings and the band at ∼1630 cm⁻¹ related to the H–O–H bending (Fig. S7†). The intensities of these peaks reduced at higher temperatures. The absorption bands characteristic for a MnO₆ core in the region 400–500 cm⁻¹ assigned to the stretching vibrations of the Mn–O bonds in manganese oxide were also observed in the FTIR spectra of these compounds. The new peak at ∼400 nm in the UV-vis spectrum of the compound can also show manganese oxide formation in this compound (Fig. S8†). The XRD was of poor resolution but it shows the layered manganese oxide in the film (Fig. 2). The layered manganese oxide was also detected by TEM (Fig. 3). The layered structure of the manganese oxide is not surprising as the method is similar to the one used to synthesise birnessite manganese oxide.⁴

Fig. 2 Powder X-ray diffraction patterns and related peaks for the layered manganese oxides in the manganese oxide–BSA film.

Fig. 3 The TEM (a, b) and HRTEM images (c, d) of the manganese oxide–BSA film.

The results show that BSA not only induces nucleation, but it could inhibit the further growth of manganese oxide as without BSA larger particles are formed. Moreover, the dispersion of manganese oxide in water is unique and may also increase the rate of reaction of particles with reactants, and could result in a high rate for many reactions.

As shown in Fig. 3, very small particles of nano-sized manganese oxide attached to the BSA protein can be observed.

In the nano-sized manganese oxide–BSA compound, manganese oxide is amorphous, and a specific phase could not be identified.

The AFM and EDX elemental map images show that nucleation occurs at special locations most probably near imidazole and carboxylate groups (Fig. 4).

Fig. 4 The AFM image shows that nucleation (white areas) occurs at special locations (a). The SEM image (b) and EDX elemental maps of the SEM image for Mn (c), N (d), C (e), O (f) and all of these elements in one image ((g) Mn: yellow, N: green, C: blue and O: red).

(NH₄)₂Ce(NO₃)₆ is a strong non-oxo transfer oxidant and one-electron oxidant. This material is widely used as the primary oxidant in the oxidation of water to oxygen catalyzed by manganese compounds because of its solubility and stability in water.^4,10 The nano-sized manganese oxide–BSA is a good catalyst for water oxidation in the presence of Ce(IV) (Fig. 5). The rate of oxygen evolution of this compound in the presence of Ce(IV) (0.1 M) is 270 μmol O₂ per mol Mn per second.

Fig. 5 Water oxidation in an aqueous solution of 0.1 M Ce(IV) at 25.0 °C in the presence of nano-sized manganese oxide–BSA solution (top). Water oxidation in an aqueous solution of 0.2 mM [Ru(bpy)₃]Cl₂ at 25.0 °C in the presence of nano-sized manganese oxide–BSA film (bottom). In the presence of BSA, without manganese oxide, no oxygen was observed.

Ru(bpy)₃⁺² has been widely applied as a photosensitizer in artificial photosystems because of its superior photophysical properties with moderate pH.¹⁰ The photoinduced oxidation of manganese oxides in the presence of [Ru(bpy)₃]⁺² in aqueous solution needs the use of a sacrificial oxidant that is irreversibly reduced allowing the net conversion of [Ru(bpy)₃]⁺² to [Ru(bpy)₃]⁺³ (Scheme 1). [Ru(bpy)₃]⁺³ can then act as an oxidant toward compounds by an intermolecular electron transfer since the potential of the [Ru(bpy)₃]⁺²/[Ru(bpy)₃]⁺³ redox couple in aqueous solution is E_1/2 = 1.1 V vs. Ag/AgCl/KCl (3.0 M).

Scheme 1 Schematic of the photocatalytic water oxidation by the [Ru(bpy)₃]²⁺ as a photosensitizer and the nano-sized manganese oxide–bovine serum albumin. A proposed mechanism for the water oxidation by nano manganese oxide–BSA. Similar to PSII, the hybrid may immobilize manganese oxides in a hydrophilic and highly oxidation stable environment (bottom).

In this study, K₂S₂O₈ was used as the electron acceptor for water oxidation during irradiation with visible light (λ > 400 nm), oxygen evolution was observed upon adding the nano-sized manganese oxide–BSA compound to an aqueous solution containing tris(2,2′-bipyridyl)ruthenium(II) chloride and K₂S₂O₈. The intensity of light was less than one-tenth of bright sunlight (on a bright sunny day illumination can be more than 100,000 lux) (Fig. 5). The rate of oxygen evolution of this compound in these conditions is 140 μmol O₂ per mol Mn per second.

Compared with the water oxidation activity of other manganese oxides nano-sized manganese oxide–BSA materials are good catalysts for water oxidation thus showing that the incorporation of manganese oxides into BSA can improve the water oxidation activity of these compounds (Table S1, ESI†). At least, BSA could help in the dispersion of these nanoparticles in solution, and the dispersivity of the particles in water could increase the rate of the reaction of the particles with oxidants (Ce(IV) or [Ru(bpy)₃]⁺³).¹⁶

As reported in previous publications,¹⁹ we propose that the O–O bond may be formed by the attack of an outer-sphere water on an OH attached to a highly valent manganese ion in the oxide structure. The attack of the water molecule on the OH group may occur by a nucleophilic, radical or radical–nucleophilic mechanism (Scheme 1).

In conclusion, we introduced BSA as a promising structural and functional model for stabilizing proteins in PSII. Both BSA and the stabilizing protein in PSII are proteins that form hydrophilic and highly oxidation stable environments for manganese oxides. It is clear that the sequence of amino acids is different in the stabilizing protein in PSII. In other words, the 3 billion year evolutionary experiments by nature have provided a better sequence of amino acids around manganese ions to form an efficient catalyst.

MMN and DJS are grateful to the Institute for Advanced Studies in Basic Sciences for financial support. CKK and SLS acknowledge the support of the US Department of Energy, Office of Basic Energy Sciences, Division of Chemical, Geological and Biological Sciences. Fig. 1 was made with VMD and the images are owned by the Theoretical and Computational Biophysics Group, NIH Resource for Macromolecular Modeling and Bioinformatics, at the Beckman Institute, University of Illinois at Urbana-Champaign. The original data for Fig. 1 is from ref. 7 (PDB: 3ARC).

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Footnote

† Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c2ra21251j

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