Photosensitized H2 generation from "one-pot" and "two-pot" assemblies of a zinc-porphyrin/platinum nanoparticle/protein scaffold

Article abstract of DOI:10.1039/c5dt03418c

We report photosensitized H₂ generation using a protein scaffold that nucleates formation of platinum nanoparticles (Pt NPs) and contains "built-in" photosensitizers. The photosensitizers, zinc-protoporphyrin IX or zinc-mesoporphyrin IX (ZnP) were incorporated in place of the naturally occurring heme in the 24-subunit iron storage protein bacterioferritin (Bfr) when the ZnPs were added to the E. coli expression medium. We engineered a stable dimeric Bfr variant with two protein subunits sandwiching a ZnP. Ten glycines were also substituted in place of residues surrounding the vinyl side of the porphyrin in order increase access of solvent and/or redox agents. An optimized "one-pot" reaction of this glycine-substituted ZnMP-Bfr dimer with a Pt(iv) salt and borohydride resulted in a ～50 : 50 mixture of protein in the form of Pt-free glycine-substituted ZnP-Bfr dimers and re-assembled 24-mers surrounding Pt NPs formed in situ. H₂ production occurred upon visible light irradiation of this "one-pot" product when combined with triethanolamine as sacrificial electron donor and methyl viologen as electron relay. An analogous "two-pot" system containing mixtures of separately prepared Pt-free glycine-substituted ZnP-Bfr dimer and porphyrin-free Pt NP@Bfr 24-mer also showed robust photosensitized H₂ generation. The glycine-substituted-ZnP-Bfr dimer thus served as photosensitizer for catalytic reduction of methyl viologen by triethanolamine, and the reduced methyl viologen was able to transfer electrons across the Bfr 24-mer protein shell to generate H₂ at the enclosed Pt NP in a "dark" reaction. Our results demonstrate that Bfr is a readily manipulatable and versatile scaffold for photosensitized redox chemistry.

Full text of DOI:10.1039/c5dt03418c

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Photosensitized H2 Generation from “One-Pot” and “Two-Pot”
Assemblies of a Zinc-Porphyrin/Platinum Nanoparticle/Protein
Scaffold
Emily R. Clark and Donald M. Kurtz Jr.*
Department of Chemistry, University of Texas at San Antonio, San Antonio, TX USA
*Address Correspondence to donald.kurtz@utsa.edu.
1

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Abstract
We report photosensitized H2 generation using a protein scaffold that nucleates formation
of platinum nanoparticles (Pt NPs) and contains “builtꢀin” photosensitizers. The photosensitizers,
zincꢀprotoporphyrin IX or zincꢀmesoporphyrin IX (ZnP) were incorporated in place of the
naturally occurring heme in the 24ꢀsubunit iron storage protein bacterioferritin (Bfr) when the
ZnPs were added to the E. coli expression medium. We engineered a stable dimeric Bfr variant
with two protein subunits sandwiching a ZnP. Ten glycines were also substituted in place of
residues surrounding the vinyl side of the porphyrin in order increase access of solvent and/or
redox agents. An optimized “oneꢀpot” reaction of this glycineꢀsubstituted ZnMPꢀBfr dimer with
a Pt(IV) salt and borohydride resulted in a ~50:50 mixture of protein in the form of Ptꢀfree
glycineꢀsubstituted ZnPꢀBfr dimers and reꢀassembled 24ꢀmers surrounding Pt NPs formed in
situ. H2 production occurred upon visible light irradiation of this “oneꢀpot” product when
combined with triethanolamine as sacrificial electron donor and methyl viologen as electron
relay. An analogous “twoꢀpot” system containing mixtures of separately prepared Ptꢀfree
glycineꢀsubstituted ZnPꢀBfr dimer and porphyrinꢀfree Pt NP@Bfr 24ꢀmer also showed robust
photosensitized H2 generation. The glycineꢀsubstitutedꢀZnPꢀBfr dimer thus served as
photosensitizer for catalytic reduction of methyl viologen by triethanolamine, and the reduced
methyl viologen was able to transfer electrons across the Bfr 24ꢀmer protein shell to generate H2
at the enclosed Pt NP in a “dark” reaction. Our results demonstrate that Bfr is a readily
manipulatable and versatile scaffold for photosensitized redox chemistry.
2

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Introduction
Efficient solarꢀdriven splitting of water: 2H2O ꢀ 2H2 + O2, using inexpensive, “green”
and “earth abundant” materials is a holy grail.1 One approach to this goal has been to assemble
systems that separately photoꢀdrive each of the two half reactions, i.e., either reduction or
oxidation of water. Numerous synthetic, biomimetic, and synthetic/biological hybrid approaches
to photosensitized reduction of water to H2 have been described.2ꢀ6 Several such examples using
8
porphyrins as photosensitizers are known.7, One of the earliest such aqueous systems,
diagrammed generically in Scheme 1, used a zinc(II)ꢀporphyrin (ZnP) as photosensitizer, a
sacrificial electron donor (SED, typically a tertiary amine), and methyl viologen (MV) as an
electron relay to colloidal platinum (Pt), where H2 production occurred.9ꢀ11 MV2+ is a well
established oxidative quencher of photoꢀgenerated Zn(II)ꢀporphyrin triplet states (3ZnP*),12, 13
and MV+ has been shown to supply reducing equivalents for generation of H2 on colloidal Pt in a
separate “dark” reaction.14 The earliest examples used synthetic ZnPs with high positive charge
in order to improve water solubility and inhibit porphyrin aggregation. ZnPs derived from
naturally occurring porphyrins, such as protoporphyrin IX, tend to aggregate in aqueous solution,
3
which quenches the ZnP*, thereby attenuating its redox efficiency. An approach to preventing
this aggregation has been to substitute Zn(II)ꢀprotoporphyrin IX and related ZnPs into heme
binding sites in proteins, thereby spatially isolating individual ZnP molecules.12, 13, 15
Scheme 1
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Platinum NPs (Pt NPs), which are frequently used as a hydrogenꢀevolving catalyst, have
been successfully incorporated into cageꢀlike proteins16ꢀ18 and in a few cases used with
exogenous metalꢀbased photosensitizers to generate H2.19ꢀ21 Bacterioferritin (Bfr) is a bacterial
iron storage protein analogous to mammalian ferritin.22 We used the Bfr from Escherichia (E.)
coli, which is the most extensively characterized and is representative of the Bfr family.
Structural and functional features of E. coli Bfr are shown in Figure 1. The protein is composed
of 24 identical 18.5ꢀkD subunits arranged as 12 headꢀtoꢀtail homodimers forming an
approximately spherical cage (Figure 1A) with an ~8ꢀnm interior cavity (Figure 1B). This cavity
can store up to 3,000 iron atoms as a ferric oxyhydroxide.23, 24 Unlike all other known ferritins,
the Bfr 24ꢀmer can stably bind up to 12 heme groups sandwiched between headꢀtoꢀtail subunit
pairs, as shown in Figure 1C. The hemes are separated from each other by ~4 nm, and the heme
irons are axially ligated by two methionine sulfurs, one from each subunit (Figure 1D), thereby
bridging the 12 dimer interfaces.25 The propionate side of the heme (Scheme 2) projects into the
interior cavity. Direct solvent access to the opposite (vinyl) side of the heme is obstructed by
surrounding surface loops (Figure 1C). However, crystal structures of several Bfrs (including
multiple crystal structures of E. coli Bfr)26ꢀ28 show that the heme vinyl side projects into a buried
pocket, which is occupied by a hydrogenꢀbonded network of water molecules (Figure S1). The
hemes may be involved in the redox chemistry of iron uptake or release, although this issue
remains to be clarified.22
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Figure 1. Structural features of the Escherichia (E.) coli Bfr 24ꢀmer (wtBfr) (A) protein
backbone viewed along a fourꢀfold rotational axis with hemes highlighted in yellow, N148
residues around one of the fourꢀfold axes highlighted in cyan, and two headꢀtoꢀtail dimer
subunits highlighted as a blue/green pair. (B) Slice through a surface rendering (oriented as in
(A)) showing the ~8ꢀnm interior cavity and hemes (yellow) embedded in the protein shell. (C)
View of a headꢀtoꢀtail homodimer with embedded heme (yellow spheres) and surface loop
regions above the vinyl side indicated. (D) View of a headꢀtoꢀtail homodimer along the heme
plane (stick rendering) showing axial methionine ligands to iron. Drawings were generated in
PyMOL (Schrödinger LLC) using coordinates from Protein Data Bank entry, 1bfr.29
Scheme 2
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We reasoned that replacing the heme with a structurally analogous ZnPs (Scheme 2)
could create a protein with “builtꢀin” photosensitizers capable of housing a Pt NP. Substitution of
the heme in Bfr with a Zn(II)ꢀchlorin photosensitizer has in fact been previously reported.30 Here
we describe “oneꢀpot” and “twoꢀpot” assemblies of a Pt NP/ZnP system using Bfr containing
either Zn(II)ꢀprotoporphryin IX (ZnPP) or Zn(II)ꢀmesoporphyrin IX (ZnMP) and their
photosensitized generation of H2.
Materials and Methods
Materials
All aqueous solutions were prepared in water that had been passed through a MilliꢀQ®
ultrapurification system (Merck Millipore, Inc.) to achieve a resistivity of 18 Mꢁ. All
manipulations involving ZnP and ZnPꢀBfrs were carried out in reduced room light. Porphyrins
were purchased from Frontier Scientific.
Syntheses of ZnP
ZnPs were prepared by metallation of MP or PP with zinc acetate dihydrate adapted from
a published method.31 70 mg of the porphyrin was dissolved in 15 mL HPLCꢀgrade
dimethylsulfoxide (DMSO). Solid zinc acetate dihydrate equimolar to the porphyrin was added,
and the solution was refluxed for 30 min. Zinc incorporation into the porphyrin was monitored
by the Soret and Qꢀband shifts in the UVꢀvis absorption spectrum. This in situ DMSO solution of
ZnP was added directly to 1 L E. coli cell cultures used for Bfr overexpression.
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Bfr overexpression and purification
The expression plasmid encoding E. coli Bfr, hereafter referred to as wtBfr (wild type
Bfr), has been described previously.32 The plasmid encoding a Bfr N148D variant, hereafter
referred to as Bfr dimer, was prepared using the QuikChange® method (Agilent Technologies)
with the wt Bfr expression plasmid as template. The plasmid encoding a sixꢀresidue Bfr variant,
N23G/L71G/Q72G/D73G/L74G/N148D, hereafter referred to as 5glyꢀBfr dimer, was
synthesized by GenScript (Piscataway, NJ) with codons optimized for expression in E. coli and
inserted into the overexpression plasmid, pT7ꢀ7 (the same parent plasmid used for expression of
wtBfr).33 E. coli BL21(DE3) transformed with the expression plasmids. These transformed
strains were grown in LuriaꢀBertani broth containing 100 mg/L ampicillin at 37°C.
Overexpression of the ZnPꢀcontaining proteins (ZnPꢀwtBfr, ZnPꢀBfr dimer, and ZnPꢀ5gly Bfr
dimer) was induced in shaking 1ꢀL cultures at 37 °C by the addition of 100 mg isopropylꢀbetaꢀDꢀ
thiogalactoside (IPTG) when OD600 of the cultures reached 0.5–0.7, followed immediately by
addition of 70 mg ZnP in 15 mL DMSO. After four hours incubation with shaking at 37 °C the
cells were harvested from the cultures by centrifugation. Porphyrinꢀfree wtBfr was
overexpressed from cultures grown in M9 minimal medium containing 100 mg/L ampicillin. 1ꢀL
cultures were incubated with shaking at 37°C until OD600 reached ~0.4, at which point, the
temperature of the cultures was lowered to 25°C. When OD600 reached ~0.6, 100 mg IPTG was
added. After overnight shaking of the cultures at 25°C, the cells were harvested by
centrifugation.
Porphyrinꢀfree wtBfr, ZnPꢀwtBfr, ZnPꢀBfr dimer and ZnPꢀ5glyꢀBfr dimer were isolated
and purified as follows. The cell pellets from six 1 L cultures were frozen at ꢀ80 °C overnight,
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then thawed and suspended in 50 mM 3ꢀ(Nꢀmorpholino)propanesulfonic acid (MOPS) pH 7.4, 5
mM MgSO4, RNAse and DNAse. The reꢀsuspended cells were lysed by sonication and cell
debris was removed by centrifugation (20,000 x g, 1 h). The supernatant was applied to a 10 mL
anionꢀexchange column (HiTrap Q X L, GE Healthcare), and the Bfr was eluted with 50 mM
MOPS, 1 M NaCl pH 7.4. The eluted protein was then applied to a 100 mL aluminum foilꢀ
covered column containing Sephacrylꢀ200 resin (GE Healthcare) and eluted with 50 mM MOPS,
0.5 M NaCl, pH 7.4. For the ZnPꢀcontaining Bfrs UVꢀvisible absorption spectra of eluted
fractions were obtained using a NanoDrop NDꢀ1000 spectrophotometer, and fractions with an
A433nm/A280nm ratio of ~ 2 were combined, concentrated, and washed with 50 mM MOPS pH 7.4.
The ZnPꢀBfrs were stored at ꢀ80°C in aluminum foilꢀcovered Eppendorf tubes.
Loading Bfrs with Pt NPs
All reactions were carried out at room temperature in solutions containing 50 mM MOPS
pH 7.4 and 0.5 µM porphyrinꢀfree wtBfr 24ꢀmer, 0.5 µM ZnPꢀwtBfr 24ꢀmer, 6 µM ZnPꢀBfr
dimer or 6 µM ZnPꢀ5glyꢀBfr dimer. Typical reaction volume was 3.5 mL. K2PtCl4 was added
from a concentrated stock solution to achieve Pt concentrations of either 0.4 mM or 0.8 mM.
After two hours incubation, sodium borohydride was added from a freshly prepared aqueous
stock solution to achieve concentrations of either 0.2 or 0.4 mM (0.5 mol borohydride per
K2PtCl4). The solutions were stirred for 60 min. Insoluble Pt in the form of a black precipitate
was removed by centrifugation at 10,000 x g for 5 min. Buffer was exchanged into 50 mM 2ꢀ(Nꢀ
morpholino)ethanesulfonic acid (MES) 0.5 M NaCl, pH 6, and excess reagents were removed by
either passage over desalting columns (EconoꢀPac 10DG column, BioꢀRad) or dialysis (Slideꢀaꢀ
Lyzer® Dialysis Cassette, Thermo Scientific).
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Quantifications and Characterizations
Protein was quantified by a 660 nm Protein Assay (Thermo Scientific Pierce). ZnP
concentrations were determined by UVꢀvisible absorption using molar extinction coefficients,
ε433 = 114 mMꢀ1cmꢀ1 for ZnPPꢀBfr dimer, ε427 = 102 mMꢀ1cmꢀ1 for ZnPPꢀ5glyꢀBfr dimer, ε424
=
111 mMꢀ1cmꢀ1 for ZnMPꢀBfr dimer, and ε415 = 96 mMꢀ1cmꢀ1 for ZnMPꢀ5glyꢀBfr dimer. These
extinction coefficients were determined from modified pyridine hemochromogens,34 which were
prepared by diluting the ZnPPꢀBfrs or ZnMPꢀBfrs with 40% (v/v) pyridine in 200 mM NaOH.
The absorption standard was a pyridine/NaOH solution of a known concentration of ZnP
prepared from a weighed quantity of commercial ZnP (Frontier Scientific, ≥95% purity by
HPLC). Nonꢀheme iron content was measured by either ferrozine assay35 or inductively coupled
plasmaꢀoptical emission spectrometry (ICPꢀOES). Pt content was determined by ICPꢀOES.
Fluorescence emission spectra of 60 µM 5glyꢀZnPPꢀBfr dimer and 60 µM ZnPPꢀBfr dimer in 50
mM MES 0.5 M NaCl, pH 6 at 502 nm were obtained using a SpectraNet fluorimeter. ZnMP
concentrations in the “oneꢀpot” samples were determined by UVꢀvis absorption
spectrophotometry of the Soret band immediately after Pt loading and also after separating the
Ptꢀloaded fractions into dimer and 24ꢀmer using sizeꢀexclusion chromatography (SEC). (SEC
dimer fractions did not contain Pt NPs, allowing for better visualization of the ZnP Soret
absorption without interference from Pt NP light scattering). SEC was performed on a Superose
6 10/300 GL column (GE) equilibrated with 50 mM MOPS 150 mM NaCl, pH 7.4. Protein
molecular weights were determined using a standard curve of carbonic anhydrase, ovalbumin,
conalbumin, apoferritin, and thyroglobulin (GE). 100 ꢀ 200 µL of ~ 0.4 mg/mL protein was
loaded onto the column and eluted at 0.4 mL/min with the same buffer while monitoring
absorbance at 280 nm. TEM was preformed on a JEOLꢀ2010F microscope operated at 200kV.
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Samples were prepared on ultrathin holey carbonꢀcoated copper grids (Ted Pella) and negatively
stained with 2% uranyl acetate.
Photosensitized reduction of MV2+
Solutions contained 2 mM MV2+, 0.1 mM TEOA, and 10 µM ZnPꢀBfr in 2ꢀ(Nꢀ
morpholino)ethanesulfonic acid (MES) pH 6. 2 mL of the solution was placed in a quartz
cuvette sealed with a screw cap and rubber septum and made anaerobic by repeated evacuation
and filling of the headspace with N2 gas via syringe needle connected to a vacuum line.
Irradiations were carried out at room temperature using a 300ꢀW halogen lamp focused through a
slide projector lens with the sample vial placed 5ꢀ10 cm from the lens. UVꢀvisible absorbance
spectra were recorded every 15 minutes on an Ocean Optics USB 2000 spectrophotometer.
Photosensitized generation of H2
All irradiated solutions contained the indicated concentrations of Pt NP and ZnPꢀ5glyꢀ
Bfr, as well as 2 mM MV2+, 0.5M NaCl, and 0.1 M TEOA in 50 mM 2ꢀ(Nꢀ
morpholino)ethanesulfonic acid (MES) pH 6. 3.5 mL of the solution was placed in a 6.5 mL
cylindrical glass screw cap vial (Chemglass), and the vials were left uncapped overnight in an
N2ꢀfilled glove box (Vacuum atmospheres Co.) at 4 °C in order remove O2. The vials were then
sealed with a rubber septum Mininert® cap and removed from the glove box. Continuous
irradiations were carried out at room temperature on stirred solutions using a 300ꢀW halogen
lamp focused through a slide projector lens with the sample vial placed 5ꢀ10 cm from the lens.
Light irradiance at the sample was measured using a Konica Minolta CLꢀ500A Illuminance
spectrophotometer. H2 production was monitored by periodic withdrawal of 100 – 250 uL of
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headspace gas into a gastight syringe and injection into a Varian CPꢀ3800 gas chromatograph
equipped with a Carboxen™ 1010 PLOT (Supelco) column and thermal conductivity detector.
The column was calibrated for amount of H2 using a standard curve prepared from a series of
solutions with varying known amounts of H2 gas (SigmaꢀAldrich) injected into buffer in the
same type of reaction vials used for photosensitized H2 generation.
Results and Discussion
Protein isolation and characterization
In addition to the E. coli wtBfr we engineered two variants. Asparagine148 surrounds the
six fourꢀfold rotationally symmetric channels of the 24ꢀmer (Figure 1A), and we found that
replacing N148 with the negatively charged aspartate side chain caused dissociation of the 24ꢀ
mer into stable homodimers (verified by SEC, see below). We refer to this N148D variant as Bfr
dimer. We created an additional variant based on the observation that the vinyl side of the heme
is buried under loop regions (Figure 1B, Figure 1C). We sought to increase solvent and/or
reagent access to the vinyl side by substituting four residues in these loops with glycines
(L71G/Q72G/D73G/L74G). We also made a fifth substitution (N23G) of a more buried residue
lying near the vinyl edge of the heme (Figure S1). Two sets of these five residues are
symmetrically disposed around each heme. For simplicity we refer to this engineered variant
containing both N148D and the five residues substituted with glycines as 5glyꢀBfr dimer. We
used the SWISSꢀMODEL web server (http://swissmodel.expasy.org/) to obtain a structural
model of the 5glyꢀBfr dimer using its amino acid sequence and the crystal structure of an
alternative E. coli hemeꢀcontaining Bfr dimer variant (PDB ID 3e2c) as template.26
A
comparison of solvent contact surfaces in the 5glyꢀBfr dimer structural model with that of the
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3e2c crystal structure (Figure 2) indicates that substitution with the ten total glycine residues on
the Bfr dimer opens a channel to the buried, waterꢀfilled pocket (Figure S1) leading from the
vinyl side of the porphyrin to the protein surface. The putative enlargement of the pocket and
channel together with greater loop flexibility expected from the ten glycine substitutions could
lead to increased solvent and/or reagent access to the vinyl side of the ZnP.
Figure 2. Solvent/protein molecular contact (“Connolly”) surfaces surrounding the heme in the
crystal structure of a Bfr dimer (PDB entry 3e2c) (left panels) and in a structural model of the
5glyꢀBfr dimer (right panels). Protein subunits are colored blue or green. Residues 23, 71ꢀ74 side
chains are highlighted in CPK colors. Heme atoms are represented as magenta spheres. “Top”
indicates views from the surface above the protein loops (indicated in Figure 1C), which
surround the vinyl side of the heme. “Side” indicates views approximately perpendicular to the
“Top” views and parallel to the heme plane with crossꢀsections through the subunits. Surface
renderings were generated in PyMOL (Schrödinger LLC). Hydrogen atoms were not included.
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The wtBfr as well as the Bfr dimer and 5glyꢀBfr dimer variants were all isolated with
essentially quantitative incorporation of ZnPP or ZnMP (~1 ZnP per dimer) when ZnP was
added to the E. coli culture medium at the time of induction of protein expression. The ZnPs
remained irreversibly bound to the wtBfr, Bfr dimer, and 5glyꢀBfr dimer under the conditions
used for the studies described here. We refer to these proteins as ZnPꢀwtBfr, ZnPꢀBfr dimer and
ZnPꢀ5glyꢀBfr dimer. The UVꢀvis absorption spectra (Figure 3) of ZnPꢀwtBfr and ZnPꢀBfr dimer
are essentially superimposable. The spectra of the 5glyꢀZnPꢀBfr dimers showed small blue shifts
in both the Soret and Q bands compared to the corresponding spectra ZnPꢀBfr dimers, indicating
some perturbation of the porphyrin environment induced by the glycine substitutions. Minor
perturbations were also evident in the fluorescence emission spectra (Figure S2).
Under the same expression conditions, a Bfr variant in which the hemeꢀligating
methionine residue was substituted with leucine (M52L) failed to bind any ZnP (Pacheco, E.,
Kurtz, D. M. Jr., unpublished). The M52L variant was previously shown not to bind heme even
though it formed a stable 24ꢀmer.25 This observation and the narrow spectral bandwidths shown
in Figure 2 are consistent with ZnP insertion specifically into the heme binding site of Bfr. The
nature of the axial ligation in these ZnPꢀBfrs is unclear. Established examples of sixꢀcoordinate
Zn(II)ꢀporphyrins are extremely rare.36 While the UVꢀvis and fluorescence spectra of the ZnPꢀ
Bfrs reported here generally resemble those of fiveꢀcoordinate ZnPs in other proteins,12, 31, 37ꢀ39
we have found no reports of a fiveꢀcoordinate, thioetherꢀligated Zn(II)ꢀporphyrin.
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Figure 3. UVꢀvisible absorption spectra of Bfr and dimer variants used in this study. Spectra
were recorded of protein solutions in 50 mM MES + 0.5 M NaCl, pH 6 at 50 µM porphyrin.
When wtBfr was expressed from cultures grown in minimal media containing no added
heme or porphyrin, the isolated Bfr showed little or no porphyrin absorption (Figure 3B), and we
refer to this protein as porphyrinꢀfree wtBfr. Nonꢀheme iron in the asꢀisolated variant Bfrs was
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usually undetectable, and that in the porphyrinꢀfree wtBfr never exceeded 3 irons per subunit.
Based on ICPꢀOES, any residual iron was apparently removed during the platinum
salt/borohydride treatment described below. A 5glyꢀBfr variant containing the native N148
formed a 24ꢀmer, but for unknown reasons did not stably bind ZnPP or ZnMP.
The photochemical behavior of the ZnPPꢀcontaining Bfrs paralleled those of the ZnMPꢀ
Bfrs. For brevity the results reported below are confined to the ZnMPꢀcontaining Bfrs. Parallel
results for the ZnPPꢀcontaining Bfrs are contained in the Supplementary Information.
Photosensitization by ZnMP-Bfrs
The UVꢀvis absorption spectral time courses in Figure 4 show that the various ZnMPꢀ
+
containing Bfrs can photosensitize reduction of MV2+ to MVꢁ by the SED, triethanolamine
+
(TEOA).12 Both the rate and extent of MVꢁ formation during 4 hours of continuous white light
irradiation was significantly higher with ZnMPꢀBfr dimer compared to ZnMPꢀwtBfr and higher
+
still using the ZnMPꢀ5glyꢀBfr dimer. The higher yield of MVꢁ using the ZnMPꢀBfr dimer in
place of ZnMPꢀwtBfr (24ꢀmer) could be explained by increased access of MV2+ and/or TEOA to
the propionate side of the ZnMP, which is oriented towards the inner cavity of the 24ꢀmer
(Figure 1C,D). The additional increase in efficiency of photosensitized MV2+ reduction upon
substitution with the ten glycine residues per ZnP is consistent with the increased exposure of the
vinyl side of the ZnP in the 5glyꢀBfr dimer structural model (Figure 2). Parallel behavior for the
ZnPPꢀcontaining proteins is shown in Figure S3.
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+
Figure 4. UVꢀvis absorption spectral time course of MV2+ reduction to MVꢁ induced by
irradiation with white light from a 300 W halogen lamp (3.8 mW/cm2) of an anaerobic solution
containing 10 µM ZnMPꢀ5gly Bfr dimer, 2 mM MV, and 0.1 M TEOA in 50 mM MES, 0.5 M
+
NaCl pH 6. Positions of MVꢁ absorption maxima at 396 nm and 606 nm are indicated. Arrows
indicate absorbance increased with time. The absorption features at ~424, 540 and 580 nm are
due to the ZnMP. Inset: A396 nm time courses for MV2+ reduction using 10µM ZnMPꢀ5gly Bfrꢀ
dimer (closed squares), ZnMPꢀBfr dimer (open circles), or ZnPPꢀwtBfr (open squares). Data
points are the average of from at least three experiments with error bars representing standard
deviations.
Pt NP incorporation
Pt NPs were incorporated into the various Bfrs by reduction in an optimized “oneꢀpot”
reaction by addition of K2PtCl4 at a mol ratio of 66 Pt/Bfr dimer followed by addition of NaBH4.
The ZnMP Soret in the 5glyꢀBfr dimer band remained intact during Pt reduction and was
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superimposed on background light scattering by the Pt NPs (Figure 5A). These solutions
remained transparent with no precipitate, whereas control reactions omitting protein resulted in a
black precipitate. After workup of the 66 Pt/ZnMPꢀ5glyꢀBfr dimer/borohydride reaction
mixtures as described in Materials and Methods, mol ratios of 43 Pt/protein dimer (~520
Pt/effective 24ꢀmer) were obtained. SEC (Figure 5B) and transmission electron microscopy
(TEM) (Figure S4A) showed that this “oneꢀpot” Pt/ZnMPꢀ5glyꢀBfr dimer preparation contained
40 – 50% of the protein as dimer and the remainder as 24ꢀmer (or 24ꢀmerꢀlike structures)
surrounding Pt NPs. Only the SEC dimer fractions showed porphyrin absorption features. The
proteinꢀencapsulated Pt NPs had a diameter range of 1.5 – 6 nm with a mean of 2.7 nm (Figure
S5A). The relatively broad SEC bands of the 24ꢀmer component can be ascribed to the
distributions of NP sizes.
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Figure 5. (A) UVꢀvis absorption spectra of 6 µM ZnMPꢀ5glyꢀBfr dimer + 0.4 mM K2PtCl4 (66
Pt per Bfr dimer) before (solid) and after (dashed) addition of 0.2 mM NaBH4 and workup (see
Materials & Methods). All reactions were carried out at room temperature in 50 mM MOPS, pH
7.4. (B) Size exclusion chromatograms (SECs) of: porphyrinꢀfree wtBfr (black, solid), ZnMPꢀ
5glyꢀBfr dimer (red, solid), postꢀworkup sample from reaction of porphyrinꢀfree wtBfr with
K2PtCl4/NaBH4 at 1600 Pt/24ꢀmer (black, dashed), or postꢀworkup sample from reaction of
ZnMPꢀ5glyꢀBfr dimer with K2PtCl4/NaBH4 at 66 Pt per/Bfr dimer (red, dashed).
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The ZnMPꢀ5glyꢀBfr dimer is, thus apparently capable of assembling into a 24ꢀmer (or
24ꢀmerꢀlike structure) when surrounding Pt NPs, which we envision occurring as diagrammed in
Scheme 3.
Scheme 3
Consistent with the interpretation in Scheme 3, analogous reactions in which the Pt salt
concentration was doubled to 133 Pt per ZnMPꢀ5glyꢀBfr dimer resulted after workup in SEC
chromatograms and TEM and showing 80 – 90% of the protein in the form of 24ꢀmers
surrounding 1.5 – 6 nm Pt NPs (Figure S6). However, this higher Pt loading was accompanied
by a substantial loss of the porphyrin Soret absorption (presumably due to degradation by the
higher borohydride/protein ratio) and higher background scattering, which rendered these
preparations unsuitable for photosensitized H2 generation (see below). Similar oligomeric
proportions and porphyrin contents were obtained from analogous reactions using ZnMPꢀBfr
dimer in place of ZnMPꢀ5glyꢀBfr dimer at 66 or 133 Pt/ZnMPꢀBfr dimer.
Analogous reaction of K2PtCl4/NaBH4 with porphyrinꢀfree wtBfr using 1600 mol Pt/mol
24ꢀmer resulted in > 90% of the protein in the form of 24ꢀmers surrounding Pt NPs with
diameters of 2.5 ꢀ 5 nm (Figure 5B, Figure S4B and Figure S5B). Pt salt/borohydride reactions
with the ZnMPꢀwtBfr resulted in significant porphyrin degradation. Attempts to incorporate
ZnPs into the porphyrinꢀfree wtBfr after Pt NP incorporation were unsuccessful.
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Photosensitized H2 generation
As shown in the “Oneꢀpot” panel of Figure 6, in the presence of TEOA and MV, we
observed photosensitized production of H2 for “oneꢀpot” preparations containing the ZnMPꢀ
5glyꢀBfr dimer in which SEC and TEM showed an ~50:50 mixture of protein as Ptꢀfree Bfr
dimer and Pt NP@Bfr 24ꢀmer. White light irradiation of “oneꢀpot” post workup samples
containing 5.3 ± 0.2 µM 5glyꢀZnMPꢀBfr dimer (estimated by protein assay and Soret absorption
of the dimer fraction collected from SEC) and 123 ± 5 µM Pt generated 0.89 ± 0.01 micromol H2
after 4 hours. No precipitation was observed before, during, or after irradiation for up to 5 hours.
Control experiments in which any one of MV2+, TEOA or Ptꢀtreated protein was omitted resulted
in no detectable photogeneration of H2. Similarly, no “dark” generation of H2 was observed in
the complete mixture. After 4 hours of irradiation, the ZnMP Soret band was no longer
detectable in the UVꢀvis absorption spectrum, which suggests that porphyrin degradation is
responsible for the leveling off of H2 generation after 4 hours. The 0.9 micromol H2 represents a
turnover of ~60 H2/ZnMP (probably a lower limit given the porphyrin photodegradation). An
analogous “oneꢀpot” preparation of ZnMPꢀBfr dimer generated no detectable H2 during four
hours of continuous irradiation (Figure 6, “Oneꢀpot” panel).
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Figure 6. Time dependence of photosensitized H2 production using either “oneꢀpot” preparations
or “twoꢀpot” mixtures. (Top panel) “Oneꢀpot” samples were from 66Pt/Bfr dimer preparations
and contained ~120 µM Pt and ~ 5 µM of either ZnMPꢀBfr dimer (closed circles) or ZnMPꢀ5glyꢀ
Bfr dimer (open circles). (Bottom panel) “Twoꢀpot” mixtures all contained porphyrinꢀfree Pt
NP@wtBfr at 20 µM Pt and either ZnMPꢀBfr dimer (closed circles), or ZnMPꢀ5glyꢀBfr dimer
(open symbols) at the indicated concentrations. All solutions contained 0.1 M TEOA, and 2 mM
MV in 50 mM MES pH 6, 0.5 M NaCl. Solutions were continuously irradiated with a 300 W
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halogen lamp (3.8 mW/cm2) at room temperature. Data points are the average from six
experiments with standard deviations indicated by vertical bars.
Based on these “oneꢀpot” results, we reasoned that the H2 production proceeded as
diagrammed in Scheme 4, in which reduction of MV2+ by TEOA was photoinduced via the
ZnMPꢀ5glyꢀBfr dimer, and H2 generation occurred in a subsequent “dark” reaction at Pt
NP@5glyꢀBfr 24ꢀmer using MV+ as the electron donor. Since the “dark” reaction at the Pt NPs
presumably does not require ZnP, we also reasoned that separately prepared porphyrinꢀfree
PtNP@wtBfr 24ꢀmer (characterized as shown in Figure 3B, Figure 5B, and Figure S4B) should
be functional in photosensitized H2 generation when mixed with ZnMPꢀ5glyꢀBfr dimer. Indeed,
continuous visible light irradiation of this “twoꢀpot” mixture in the presence of MV and TEOA
under anaerobic conditions resulted in robust photosensitized production of H2 (Figure 6, “twoꢀ
pot” panel). 7 hours irradiation with 10 µM of 5glyꢀZnPꢀBfr dimer mixed with porphyrinꢀfree Pt
NP@wtBfr 24ꢀmer at 20 µM Pt generated ~1 micromol H2. This yield represents a turnover of
~33 H2/ZnMP. Doubling the concentration of ZnMP to 20 µM approximately doubled the
amount of photoꢀinduced H2 produced after 7 hours. Loss of the porphyrin Soret absorption was
significantly slower in the “twoꢀpot” system compared to the “oneꢀpot” system, and the Soret
band was still prominent after 7 hours irradiation. Similar “twoꢀpot” H2 production was obtained
using ZnPPꢀ5glyꢀBfr dimer in place of ZnMPꢀ5glyꢀBfr dimer (Figure S7). As was the case for
the “oneꢀpot” system, no H2 was detected in “twoꢀpot” mixtures using asꢀisolated ZnPꢀBfr dimer
in place of ZnPꢀ5glyꢀBfr dimer during 4 hours of irradiation.
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Scheme 4
Conclusions
Our “twoꢀpot” results thus support the photosensitized H2 generation pathway shown in
Scheme 4 that we surmised for the “oneꢀpot” system. Photosensitization occurred via a ZnPꢀBfr
dimer modified to increase the exposure of the porphyrin vinyl side. This modification
apparently increased the efficiency of photoꢀinduced electron transfer events. “Twoꢀpot”
mixtures containing separately prepared Ptꢀfree ZnPꢀ5glyꢀBfr dimer and porphyrinꢀfree Pt
NP@wtBfr 24ꢀmer exhibited higher H2 yields and turnover frequencies per Pt, as well as more
sustained H2 generation than the optimized “oneꢀpot” mixtures. However both systems exhibited
turnover frequencies comparable to those of other systems operating according to Scheme 1
using proteinꢀbound porphyrins combined with synthetic polymerꢀcoated Pt NPs.12, 13, 15 In fact,
we obtained yields of H2 comparable to those in our “twoꢀpot” system when we substituted
poly(vinyl)alcoholꢀcoated Pt NPs40 in place of porphyrinꢀfree Pt NP@wtBfr at approximately the
same Pt concentration (data not shown). “Dark” electron transfer from external MV+ across the
wtBfr protein shell to the internalized Pt NPs is, therefore, apparently sufficient to support H2
generation according to Scheme 4. Our results demonstrate a unique proteinꢀbased
photosensitizer/Pt NP system that is easily assembled and readily modifiable for photosensitized
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redox chemistry. We are in the process of exploring additional amino acid substitutions and other
modification to the ZnP environment and to the Bfr proteins shell that could improve and expand
the photochemical performance of this novel system.
Acknowledgments
This research was supported by a grant from the American Chemical Society Petroleum
Research Fund (#49201ꢀND4 to D.M.K.). E. Clark received financial support from a University
of Texas Board of Regents Graduate Program Initiative grant. We thank E. Pacheco for the
experiments on the M52L Bfr variant, Professor Heather Shipley in the UTSA Department of
Civil and Environmental Engineering for access to and assistance with inductively coupled
plasmaꢀoptical emission spectrometry, and Judith M. Nocek for helpful discussions.
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Graphical Abstract
Photosensitized H2 generation can be sustained for several hours in aqueous solution using a
protein scaffold that nucleates formation of platinum nanoparticles (Pt NPs) and contains “built-
in” zinc-porphyrin photosensitizers.