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The goal of this project is to photo-drive reduction of H⁺ to H₂ in aqueous solutions using a protein scaffold surrounding a noble metal nanoparticle (NP) and a photosensitizing heme-analog, such as zinc-protoporphyrin IX (ZnPPIX), embedded within the protein shell.  A “sacrificial” electron donor (triethanolamine) would function as the reducing agent for H⁺. The highly reducing photo-excited triplet state of ZnPPIX would reduce H⁺ to H₂ on the surface of the internalized NP, and the resulting oxidized ZnPPIX would be re-reduced in a “dark” reaction by the sacrificial electron donor. The protein scaffold is an approximately spherical 24-subunit protein called bacterioferritin (Bfr). This protein contains an ~8-nm interior cavity and 12 identical hemes, each of which bridge two subunits and project through the protein shell.  The native function of Bfr is to store up to 3,000 non-heme iron atoms as a ferric oxyhydroxide polymer within the 8-nm cavity.

During the first year of the grant period, we succeeded in replacing the heme in Bfr quantitatively with either ZnPPIX or with another closely related and well-established photosensitizer, Zn-chlorine6. This ZnPPIX-Bfr or Znchlorine6-Bfr construct contained ~300 non-heme iron atoms within the internal cavity (Fe_~300-ZnPPIX or Znchlorine6-Bfr). Unless otherwise noted, all subsequent manipulations were conducted in low light conditions, and the proteins were stored in the dark. As a test of the photochemical reduction method, we demonstrated that irradiation of the Fe_~300-ZnPPIX-Bfr with visible light from a tungsten halogen projector lamp in the presence of the sacrificial electron donor, triethanolamine, caused reduction of the internal non-heme Fe(III) to Fe(II). The Fe(II) then diffused out through pores in the protein shell, where it was detected and quantitated by a well-established chelation/colorimetric method. In multiple experiments we were able to photo-drive reduction and release of 60-90% of the internal non-heme iron. No reduction of Fe(III) above background occurred in the absence of the visible light irradiation.

 We next removed all of the non-heme iron from the ZnPPIX-Bfr by a well-established chemical method to obtain apo-ZnPPIX-Bfr. (We did not use the photochemical reduction method for iron removal in order to avoid the possibility of photochemical damage to the ZnPPIX or the protein).  We then followed a published procedure for formation of Pt NPs in the vacated internal cavity of the ZnPPIX-Bfr. The method involves incubation of the apo-ZnPPIX-Bfr with excess PtCl₄^2-, during which the PtCl₄^2- diffuses into the protein cavity. After several hours incubation at room temperature any non-internalized PtCl₄^2- was rapidly removed from the protein solution by spin-filtration. Immediately afterwards, sodium borohydride was added to reduce the internalized PtCl₄^2- to Pt(0). Excess reagents were removed by passage over a small size-exclusion column. Transmission electron microscopy showed that some of the protein molecules treated in this manner enclosed what appeared to be electron dense particles, which were not present in protein that had not been exposed to any platinum. 

 During the next year of the grant period, our plans are to optimize the Pt NP incorporation procedure. We have introduced cysteine residues onto the walls of the internal cavity of Bfr. This sulfur-containing amino acid should increase the affinity for Pt, and possibly nucleate NP formation. By introduction of published mutations into Bfr, we have constructed an essentially apo-ZnPPIX-Bfr, which avoids the necessity of non-heme iron removal. Once the Pt NPs are incorporated into the apo-ZnPPIX- or apo-Znchlorine6-Bfr, we will attempt to detected photochemically generated H₂ by gas chromatography.