Biomolecular Catalysis - American Chemical Society

C h a p t e r 17

Biomimetic Synthesis of an Active H Catalyst Using the Ferritin Protein Cage Architecture 2

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Zachary Varpness , Chad Shoopman , John W. Peters , Mark Young , and Trevor Douglas * Downloaded by GEORGETOWN UNIV on August 19, 2015 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch017

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Departments of Chemistry and Biochemistry, Plant Sciences, and Center for Bio-Inspired Nanomaterials, Montana State University, Bozeman, MT 59717 Corresponding author: [email protected]

The possibility of developing hydrogen gas as an environmentally friendly alternative fuel has received considerable attention . However, the majority of hydrogen produced for energy yielding applications is generated by the process of reforming methane or fossil fuels . Thus, we are not in a position to develop a hydrogen energy economy that is independent of nonrenewable fossils fuels. A renewable source for the production of hydrogen is needed for the development of hydrogen as an alternative fuel. We have taken a biomimetic synthetic approach to the synthesis of efficient catalyst systems for H formation. We have combined the advantages of biological control and the strengths of synthetic chemistry to create novel materials with tailored catalytic properties. A major goal of biomimetic chemistry is to couple biological molecules with synthetic capacity to create new functional materials showing dramatic property enhancements. By interfacing hard inorganic materials (metals) with soft biological materials (proteins), we can create new composite materials with controlled multifunctionality. With the high cost and limited supply of platinum, it is necessary to explore ways of maximizing the catalytic efficiency of Pt on a per atom basis in order to develop economically feasible catalysts. In a particle based approach toward developing a Pt catalyst, it is necessary to minimize the diameter of the particle and thus increase the surface area (i.e. the number of exposed Pt atoms per particle). A number of different synthetic approaches have been used to synthesize platinum nanoparticles using different passivating layers . 1-6

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© 2008

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In Biomolecular Catalysis; Kim, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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264 However, the passivating layers generally interfere with the exposed Pt atoms and reduce catalytic efficiency . In this study, we have employed the protein cage of ferritin as a synthetic platform, which, unlike a passivating layer, does not coat the entire surface of the nanoparticle but still isolates the nanoparticle in solution and prevents aggregation. We have previously demonstrated that protein cage architectures can be used as bio-templates for the synthesis of encapsulated nanomaterials " . Protein cage architectures are structurally-defined, container-like assemblies. They are self-assembled from a limited number of protein subunits in which the interior and exterior surfaces are chemically distinct. Through genetic and chemical modifications, reaction sites can be inserted into specific locations within the cage architecture to create designed functionality . Protein cages have been used to not only to define the size and shape of nanomaterials but also the crystal phase . We have adopted this biomimetic approach to create a ferritin-based catalyst for the production of H . Ferritin is a remarkably stable protein architecture that assembles from 24 subunits into a 12 nm cage defining an 8 nm interior cavity (Figure 1). Ferritin is an iron storage protein found a nearly all organisms and 9

Downloaded by GEORGETOWN UNIV on August 19, 2015 | http://pubs.acs.org Publication Date: June 23, 2008 | doi: 10.1021/bk-2008-0986.ch017

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Figure 1. (A) Space filling representation of the horse spleen ferritin cage (pdb: 1AEW). (B) Cut-away view offerritin showing the interior cavity of the cage.

has been used as a platform for the synthesis of a range of nanoparticles, including catalysts such as palladium " . We have previously shown that catalytically active platinum nanoparticles can be synthesized inside another protein cage, the small heat shock protein from Methanococcus jannaschii . In the current study, we have demonstrated synthesis and activity of Pt nanoparticles within the ferritin protein cage architecture. 26

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265 The synthesis of ferritin encapsulated Pt nanoparticles was undertaken using synthetic methodology similar to previously reported . Briefly outlined, demineralized and purified apo-ferritin (5 mg, 0.01 ^moles) was incubated with either 250 or 1000 PtCl " per protein cage at 65°C for 15 minutes. Reduction with dimethylamine borane complex ((CH ) NBH ) resulted in the formation of a brown colored solution. Characterization of the reaction product by size exclusion chromatography revealed retention volumes identical to untreated ferritin and showed co-elution of protein (280 nm) and Pt (350 nm) components (Figure 2). Dynamic light scattering indicated no change in the particle 25

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Figure 2. Size exclusion chromatography of ferritin. (A) Unmineralized ferritin, (B) Ferritin mineralized with 1000 Pt/ferritin showing coelution of protein (280 nm) and mineral (350 nm), and (C) Ferritin mineralized with 250 Pt/ferritin showing coelution of protein (280 nm) and mineral (350 nm).

diameter after the reaction. Transmission electron microscopy (TEM) of the Pttreated ferritin (Fn-Pt) revealed homogeneously sized electron dense cores 1.9 ± 0.8 nm in diameter (Figure 3A). These particles were identified as Pt metal by electron diffraction as a powder pattern was observed (Figure 3A, inset) with the d-spacings shown in Table I. Negatively stained TEM samples displayed 12 nm protein cages with Pt particles clearly localized within the cage structure (Figure 3B). For theoretical loadings of 1000 Pt/cage, nanoparticles of 1.9 ± 0.8 nm were observed (Figure 3C) and for loadings of 250 Pt/cage, particles of 1.8 ± 1.2 nm were observed (Figure 4). Ferritin-free control reactions resulted in the formation of aggregated Pt colloids, which rapidly precipitated from solution. Control reactions using bovine serum albumin (BSA), at the same total protein concentration, also resulted in bulk precipitation and only a fraction of the Pt


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Table I. Measured d-spacings Miller Index

d-spacing

Measured d-spacing

111 200 220 113

2.265 1.961 1.387 1.183

2.304 1.965 1.140 1.195

Figure 3. (A) TEM of ferritin 1000 Pt unstained. The inset shows electron diffraction of Pffrom ferritin 1000 Pt. (B) TEM of ferritin 1000 Pt stained with 2% uranyl acetate. (C) Histogram of Pt particle diameters in ferritin 1000 Pt. Average 1.9 ± 0.8 nm from 215 particles. Scale bar = 50 nm.



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Figured (A) TEM of ferritin 250 Pt stained with 2 % uranyl acetate. (B) Histogram of Pt particle diameters in ferritin 250 Pt. Average 1.8 ± 1.2 nm from 99 particles. Scale bar = 50 nm.

Figure 5. BSA mineralization control reaction. Scale Bar = 200 nm.


268 remained in solution with a wide distribution of particle sizes (3 nm to 120 nm) when observed by TEM (Figure 5). The Pt-Fn protein cage composites are highly active catalysts able to reduce H to form H when supplied with an appropriate reducing agent. We have used visible light and a co-catalyst (Ru(bpy) ) to generate MV* through oxidation of simple organics such as EDTA (Figure 6) to assay H production from the Fn-Pt catalyst. In this assay, the solution was illuminated at 25°C with a 150W Xe-arc lamp equipped with an IR filter and a UV cut-off filter (360 nm). The Pt-Fn (0.5 /ig, 1.1 xlO" moles) was illuminated in the presence of MV (0.5 mM), Ru(bpy) (0.2 mM) and EDTA (200 mM) at pH 5.0 and the resulting H was quantified by gas chromatography (Shimadzu GC-8A). +

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Figure 6. Schematic of the light mediated H production from Pt-Fn. Methyl viologen (MV ) is used as an electron transfer mediator between the Rufbpy)/ photocatalyst and the Pt-Fn responsible for H production. 2

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The initial rates of H formation, when calculated on a per cage basis, were 14.9 x 10 H /sec/cage (± 2.77 x 10 H /sec) for a loading factor of 1000 Pt per ferritin (Figure 7) and 1.82 x 10 H /sec/cage (± 8.47 x 10 H /sec) for a loading factor of 250 (Figure 8). When the H production rates are calculated on a per Pt basis, they compare very favorably with other reported Pt nanoparticles. The initial rates for Pt-Fn with 1000 Pt/cage are 893 H /Pt/min, as compared with the reported literature values (20 H /Pt/min , 16 H /PtAnin , and 6.5 H /Pt/min ). In addition, initial H production rates for Pt-Fn are more active than the previous reports using Pt nanoparticles synthesized inside of small heat shock protein (Pt-Hsp). The initial rates of H production from Pt-Hsp with 1000 Pt/cage are 268 H /Pt/min . The Pt-Fn synthesis reaction produced Pt particles with the size of 1.9 ± 0.8 nm as compared to particles of 2.6 ± 0.2 nm for previous synthesis in Hsp under the same conditions. The particular protein cage 2

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269 platform employed in the synthesis therefore has a significant effect on the size and activity of the catalyst nanoparticle. After 20 minutes of illumination, the Ru(bpy) begins to photobleach and the methyl viologen undergoes platinum catalysed hydrogenation, which significantly decreases the hydrogen production. The smaller size of the particles in Pt-Fn compared to Pt-Hsp can, 2+


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Figure 7. H production from 1000 Pt/Ferritin in 0.2 mM Ru(bpy) , 0.5 mM methyl viologen, 200 mM EDTA, and 500 mM acetic acid pH 5.0. • 1000 Pt/ferritin 1.1 x 10' moles Pt. 2

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Figure 8. H production from 250 Pt/Ferritin in 0.2 mM Ru(bpy) , 0.5 mM methyl viologen, 200 mM EDTA, and 500 mM acetic acid pH 5.0. • 250 Pt/ferritin 3.3 x 10' moles Pt. 2

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270 in part, explain the increased H production. Based on the particle size histograms from Pt-Fn and Pt-Hsp, it appears that Pt-Fn has a narrower size distribution compared to Pt-Hsp. This distribution suggests that the Pt-Hsp will have more particles that are in the range too small to be detected by TEM, which are inactive catalysts. Pt-Fn has fewer particles that are below the threshold of detection giving rise to an overall more active preparation. Pt-Fn could more efficiently mineralize catalytically active platinum particles by making more particles over the catalytically active threshold of 50 atoms of platinum while Pt-Hsp has more platinum in particles that are inactive lowering the per Pt eflficency. We have used a well-defined thermally stable protein cage architecture to synthesize an encapsulated catalyst for producing H . We have introduced nanoscale metal clusters, in a spatially selective manner, to the interior of the ferritin protein cage architecture. The encapsulated Pt nanoparticles act as active sites for the reduction of H to form H , and the activity of these bio-composite materials are significantly better than previously described Pt nanoparticles. The protein cage architecture of ferritin prevents the agglomeration of the nanoparticles while minimizing effects of passivating the surface of the catalyst. The Pt-Fn composite illustrates the utility of using protein architectures for the formation of functional nanomaterials with applications in energy production and utilization. 2


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Acknowledgements This work was funded in part by grants from the Office of Naval Research (19-00-R0006). Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).

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Biomolecular Catalysis - American Chemical Society

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