Photosensitized H2 Production Using a Zinc Porphyrin-Substituted

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Photosensitized H2 Production Using a Zinc Porphyrin-Substituted Protein, Platinum Nanoparticles, and Ascorbate with No Electron Relay: Participation of Good’s Buffers Emily R. Clark and Donald M. Kurtz, Jr.* Department of Chemistry, University of Texas at San Antonio, San Antonio, Texas 78249, United States S Supporting Information *

ABSTRACT: Development of efficient light-driven splitting of water, 2H2O → 2H2 + O2, often attempts to optimize photosensitization of the reductive and oxidative half-reactions individually. Numerous homogeneous and heterogeneous systems have been developed for photochemical stimulation of the reductive half reaction, 2H+ + 2e− → H2. These systems generally consist of various combinations of a H+ reduction catalyst, a photosensitizer (PS), and a “sacrificial” electron donor. Zinc(II)-porphyrins (ZnPs) have frequently been used as PSs for H2 generation, but they are subject to various self-quenching processes in aqueous solutions. Colloidal platinum in nanoparticle form (Pt NP) is a classical H+ reduction catalyst using ZnP photosensitizers, but efficient photosensitized H2 generation requires an electron relay molecule between ZnP and Pt NP. The present report describes an aqueous system for visible (white) light-sensitized generation of H2 using a protein-embedded Zn(II)-protoporphyrin IX as PS and Pt NP as H+ reduction catalyst without an added electron relay. This system operated efficiently in piperazino- and morpholino-alkylsulfonic acid (Good’s buffers), which served as sacrificial electron donors. The system also required ascorbate at relatively modest concentrations, which stabilized the Zn(II)-protoporphyrin IX against photodegradation. In the absence of an electron relay molecule, the photosensitized H2 generation must involve formation of at least a transient complex between a protein-embedded Zn(II)-protoporphyrin IX species and Pt NP.

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oxidative quenching of 3ZnP* by oxidized methyl viologen (MV2+) and subsequent reduction of the ZnP•+ by the SED, ethylenediaminetetraacetic acid (EDTA), as shown in Scheme 1. The reduced methyl viologen (MV•+) served as electron

INTRODUCTION A widely used approach to develop efficient light-driven splitting of water, 2H2O → 2H2 + O2, under mild conditions, is to optimize systems that drive each of the two half-reactions individually. Numerous homogeneous and heterogeneous systems have been developed for photochemical stimulation of the reductive half reaction 2H+ + 2e− → H2.1−11 These systems generally consist of various combinations of a H+ reduction catalyst (colloidal platinum, transition-metal complexes, hydrogenase, or hydrogenase mimic), a photosensitizer (PS) (molecules with relatively low-lying and long-lived triplet excited states, natural photosynthetic reaction centers, or semiconductors), a “sacrificial electron donor” (SED, most often tertiary amines, thiols, or ascorbate) and, if necessary, an electron relay molecule (most often, methyl viologen) between PS and catalyst. Many of these systems suffer from photoinstability, pH extremes, or a requirement for solubilizing organic solvents. Although development of H+ reduction catalysts using earth-abundant materials has been an understandable goal, colloidal platinum continues to be used due to its ease of synthesis and manipulation, durability, and catalytic efficiency. Zinc(II)-porphyrins (ZnPs) have been frequently used as PSs for H2 generation.12,13 The first reported aqueous ZnP system utilized the water-soluble zinc(II)-tetra(Nmethylpyridyl)porphyrin (ZnTMPyP) as PS and a platinum nanoparticle (Pt NP) catalyst.14,15 This system operated via © 2017 American Chemical Society

Scheme 1. Photosensitized H2 Generation Using ZnP, EDTA, MV, and Pt NP

relay to Pt for catalytic H2 production, and the system required a large excess of MV to function efficiently. In the absence of MV, reductive quenching of the 3ZnP* to the Zn(II)-porphyrin anion radical (ZnP•−) by EDTA was observed. In the presence of Pt NPs, this reductive quenching pathway also produced H2, apparently by direct electron transfer from the ZnP•− to Pt, but in much lower yields and only at much higher Pt concentrations than in the presence of MV.15,16 This reductive Received: January 25, 2017 Published: March 31, 2017 4584

DOI: 10.1021/acs.inorgchem.7b00228 Inorg. Chem. 2017, 56, 4584−4593

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Inorganic Chemistry

Figure 1. Relevant structural features of the E. coli Bfr (A) 24-mer backbone assembly with hemes highlighted in yellow; 4 of the 24 N148 residues that were changed to D are highlighted in cyan, and two head-to-tail dimer subunits are highlighted as a blue/green pair. (B) View of a head-to-tail homodimer approximately perpendicular to the plane of the embedded heme (CPK colored spheres). The heme propionate groups protruding from the left side are oriented toward the interior of the protein shell. (C) Close-up view of a head-to-tail homodimer along the heme plane (stick rendering) showing axial methionine ligands to iron. (D) Heme. Protein drawings were generated in PyMOL (Schrödinger LLC) using coordinates from Protein Data Bank entry 1bfr.22

quenching also led to ZnP•− disproportionation and porphyrin degradation. In aqueous solution, aggregation of less water-soluble ZnPs derived from naturally occurring porphyrins such as protoporphyrin IX (PP) greatly lowers their efficiencies as PSs. One approach to preventing direct interactions between Zn(II)protoporphyrin IX (ZnPP) molecules has been to embed them within heme binding sites of proteins.17,18 We previously reported a one-pot assembly of a photocatalytic aqueous system for H2 generation using a ZnPP-substituted Escherichia coli bacterioferritin (Bfr) as PS.19 Bfr is a 24-subunit iron storage protein with the unique ability among ferritins to stably bind hemes at specific sites in its protein shell, as shown in Figures 1A−C.20,21 ZnPP is isostructural with heme (Figure 1D), except that Zn(II) is substituted in place of Fe(II). We showed that ZnPP could be quantitatively substituted in place of heme simply by adding ZnPP to the Bfr expression cultures. We further showed that an engineered charge change variant, N148D (Figure 1A), caused dissociation of the 24-mer into dimers, in which one ZnPP is sandwiched between two identical protein subunits (as shown for heme in Figures 1B and C). This N148D ZnPP-Bfr did not support photosensitized H2 generation in a system operating according to Scheme 1. Photosensitized H2 generation was observed only upon five additional amino acid residue substitutions on N148D Bfr, which generated a ZnPP-Bfr dimer variant capable acting as PS. We attributed the photosensitizing ability of this six-residue variant to increased access of the SED and/or MV to the ZnPP.19 The methionine residue supplying the axial heme ligands (Figure 1C) was not among the substituted residues. The axial ligation state of the Zn(II) in these ZnPP-Bfrs has not been established. E. coli Bfr contains no cysteine residues, which could conceivably participate in photosensitized electron transfer or quenching, and a cysteine residue was not introduced in the six-residue variant. We also reported that Pt NPs could be loaded into the hollow interior cavity of the porphyrin-free wtBfr 24-mer (pfwtBfr). This Pt NP@pf-wtBfr was used as the catalyst for photosensitized H2 generation in combination with the sixresidue variant ZnPP-Bfr dimer as PS and MV as electron relay.19 Analogous loading of Pt NPs into ZnPP-wtBfr resulted in substantial ZnPP degradation, and this porphyrin-degraded Pt NP@Bfr 24-mer did not support photosensitized H2 generation when combined with MV and SED.

Efficient porphyrin-photosensitized H2 production using Pt NPs in aqueous solution has proven difficult to achieve without a large excess of a viologen and to our knowledge has not been previously reported for the aggregation-prone free ZnPP or related biological porphyrins. The present report describes an aqueous system for sustained photosensitized generation of H2 using the single-residue variant N148D ZnPP-Bfr as PS and Pt NPs as H+ reduction catalyst without a viologen or any other added electron transfer mediator. Photosensitized H2 generation was achieved in piperazino- and morpholino-alkylsulfonic acid Good’s buffers23,24 but not in phosphate buffer or high salt solutions and also required ascorbate at relatively modest concentrations.

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EXPERIMENTAL SECTION

Reagents and General Methods. All chemicals were purchased at the highest available purities. Protoporphyrin IX (PPIX) and zinc(II)-meso-tetra (N-methyl-4-pyridyl) porphyrin (ZnTMPyP) were purchased from Frontier Scientific. Sodium L-ascorbate, 3-morpholinopropane-1-sulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium borohydride, trisodium citrate, and disodium EDTA were purchased from either Fisher Scientific or Sigma-Aldrich. ZnPP was prepared by metalation of PPIX with zinc acetate dihydrate.19 All aqueous solutions were prepared in water that had been passed through a Milli-Q ultrapurification system (Merck Millipore, Inc.) to achieve a resistivity of 18 MΩ. Expression, isolation, and purification of pf-wtBfr and N148D ZnPP-Bfr have been reported previously.19 All cell cultures and proteins containing ZnPP were stored and manipulated in a low light environment or aluminum foil-covered containers. ZnPP and metal contents in the purified N148D ZnPP-Bfr and pf-wtBfr were quantified as described previously.19 Separation of N148D ZnPP-Bfr Dimer from 24-mer. Oligomerization states of the various Bfrs were determined by size exclusion chromatography (SEC). The purified protein was loaded in 1 mL increments onto aluminum-foil covered Superose 6 10/300 GL column (GE Healthcare) packed with 24 mL of cross-linked agarose. Protein was eluted at 0.4 mL/min with 50 mM MOPS and 150 mM NaCl (pH 7.4) sterilized by 0.22 μm filter. Absorbance was monitored at 280 nm. Dextran Blue was used to determine the void volume of the column. A standard curve was obtained using ovalbumin (43 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) (GE Healthcare). ZnPP-wtBfr was used as the standard for the 24-mer, and N148D ZnPP-Bfr was used as the standard for the dimer. Fractions that eluted at a time corresponding to the Bfr dimer (37 kDa) were collected, exchanged into ultrapure H2O, and concentrated to 0.3−0.5 mM. Typical yields of protein were 4585

DOI: 10.1021/acs.inorgchem.7b00228 Inorg. Chem. 2017, 56, 4584−4593

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Inorganic Chemistry ∼5 mL of 0.3 mM Bfr (concentration expressed as dimer units). After purification and sizing, Bfr was stored as 500 uL aliquots in 1.5 mL aluminum foil-covered Eppendorf tubes at −80 °C. Preparation and Characterization of Pt NPs. Citrate-coated Pt NPs (Pt NP@citrate) were prepared essentially as described by Wu et al.25 One milliliter of an aqueous 16 mM H2PtCl6 solution was allowed to hydrolyze for ≥3 h before being combined with 1 mL of an aqueous 40 mM trisodium citrate solution. The H2PtCl6/citrate solution was diluted to a total volume of 40 mL with water to achieve concentrations of 0.4 mM Pt and 1 mM citrate, and the diluted solution was vigorously stirred for ≥1 h. Sodium borohydride was then added from a freshly prepared aqueous stock solution to achieve a concentration of 0.5 mM. The solution was stirred for 3 h. A color change from yellow to brown indicated formation of Pt NPs. The solutions were washed with water by repetitive concentrations and dilutions using 10 kDa MWCO Amicon centrifugal filter units (Millipore). Approximately 6 mL of 1.5 mM Pt as Pt NPs were typically obtained. Pt NP@pf-wtBfr was prepared as previously described.19 Pt concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) from 100 μL of Pt NPs solutions prepared as described above, diluted to 500 μL with freshly prepared aqua regia, and then further diluted to 5 mL with water. Pt NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) at the University of Texas at San Antonio Department of Physics and Astronomy. For TEM, 10 μL of Bfr samples containing ∼40 μg/mL Pt were placed on ultrathin holey carbon-coated copper grids (Ted Pella) and allowed to dry at room temperature. Samples were run on a JEOL 2010F field emission transmission electron microscope at 200 kV. DLS sizing and ζpotentials were measured on a Malvern Instruments Zetasizer Nano ZS at the Department of Physics and Astronomy at the University of Texas at San Antonio. For DLS sizing, 1.5 mL samples of ∼6 μM Pt as Pt NPs and/or ∼3 μM ZnPP-Bfr were prepared in polystyrene cuvettes (Fisher Scientific). ζ-potentials were measured at the same Pt and/or protein concentration as that for DLS using 1 mL DTS1070 folded capillary cells with attached electrode (Malvern). ZnPP-Bfr Photosensitized Generation of H2. Stock solutions of 100 mM sodium ascorbate, 100 mM MV2+, and 500 mM disodium EDTA were prepared in 1.5 mL of ultrapure H2O in Eppendorf tubes, then frozen at −80 °C. Frozen reagent and N148D ZnPP-Bfr stock solutions were removed from the −80 °C freezer and brought into the antechamber of a N2-filled Vacuum Atmospheres Co. glovebox along with degassed buffer, 500 μL of Pt NP@citrate solution in closed Eppendorf tubes, and 6.5 mL cylindrical glass screw cap vials (Chemglass) with rubber septa (Mininert). The antechamber atmosphere was cycled three times between vacuum and N2 before items were brought into the glovebox. Solutions were left uncapped in the glovebox for 45−60 min to thaw and equilibrate with the N2 atmosphere. N2 was repeatedly bubbled into the Pt NP solutions via pipet. All other reagents were degassed on the Schlenk line by repetitive evacuation/N2 cycles before being brought into the glovebox. These stock solutions and anaerobic buffer solutions in capped tubes were stored in a 4 °C mini-refrigerator in the glovebox. Ascorbate and N148D ZnPP-Bfr stock solutions were used within one week. Pt NP@citrate preparations were found to have a functional lifetime of up to 30 days. Pt NP@pf-wtBfr preparations were used within 14 days. Solutions for irradiation were prepared and maintained under anaerobic N2 atmosphere. Concentrated reagent stock solutions were added to the 6.5 mL vials, and the mixtures were diluted to 3 mL final volume with anaerobic buffer. The vials were then capped and removed from the glovebox. Continuous white light irradiations of solutions were carried out essentially as described previously19 at room temperature (∼23 °C) using a 300 W halogen lamp focused through a slide projector lens with a HOYA 62 mm UV-IR multi-coated filter with wavelength cutoffs below 390 nm and above 700 nm. H2 production was monitored by gas chromatography, essentially as described previously.19 Detailed procedures for headspace sampling and for calibration of the gas chromatograph are described in the

Supporting Information. Protein, reagent, and buffer compositions in the irradiated solutions are listed in the figure legends. Error bars on data points represent standard deviations from the average from at least three samplings. UV−Vis Absorption Spectra of Irradiated Solutions. Solutions were prepared and irradiated as described above for H2 sampling except using 2 mL volumes in 3 mL 1 cm path length quartz cuvettes (Starna) with screw top caps and PTFE septa. Spectra were obtained using an Ocean Optics USB 2000 spectrophotometer. ZnTMPyP Photosensitized Generation of H2. ZnTMPyP (1.3 mg) was dissolved in 1.47 mL ultrapure H2O in an Eppendorf tube to achieve a 1 mM stock solution, which was used within 24 h of preparation. The closed Eppendorf tube was brought into the antechamber of the N2-filled glovebox. The antechamber atmosphere was cycled three times between vacuum and N2 before ZnTMPyP was brought into the glovebox. ZnTMPyP solutions were uncapped, and N2 atmosphere was repeatedly bubbled through the solution via pipet to remove dissolved O2. Other reagents were prepared and manipulated as described above for the N148D ZnPP-Bfr experiments. Concentrations, solution and headspace volumes, photosensitized H2 generation procedure, and headspace sampling procedure were identical to those used for the N148D ZnPP-Bfr solutions.

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RESULTS Characterization of Pt NPs. Pt NPs with two different coatings were used. TEM showed that Pt NP@citrate preparations contained well-dispersed, approximately spherical Pt NPs with diameters of 2.5−3 nm (Figure S1 in the Supporting Information). As described previously,19 Pt NPs in unstained TEM images of Pt NP@pf-wtBfr had diameters of 1.5−5 nm, and uranyl acetate-stained TEMs showed the expected 10−12 nm Bfr 24-mer protein shell surrounding the Pt NPs.26 Corresponding DLS characterizations (Figure 2)

Figure 2. DLS of Pt NP@citrate (solid trace) and Pt NP@pf-wtBfr (dashed trace). Both solutions contained ∼6 μM Pt as Pt NPs in 50 mM MOPS, pH 7.4.

were consistent with the TEM results, showing mode diameters of 2.7 nm for Pt NP@citrate and 10.1 nm for Pt NP@pf-wtBfr. ζ-potentials were −30 ± 12 mV for Pt NP@citrate and −9.1 ± 0.8 mV for Pt NP@pf-wtBfr. These values reflect the more concentrated negative surface charge expected for the Pt NP@ citrate. N148D ZnPP-Bfr ZnPP and Metal Contents. As described previously,19 the N148D ZnPP-Bfr used in this work contained 1 ZnPP/Bfr dimer and 1 mM N148D ZnPP-Bfr on a protein dimer basis contained 60−90% of the protein in 24-mers. Diluting these concentrated Bfr solutions to 0.1−0.5 mM (dimer basis) and incubating them for 24 h at 4 °C shifted the oligomer proportions toward the dimer, resulting in 90−100% dimer and 0−10% 24-mer. Reconcentrating the protein resulted in a shift back toward 24-mer over the course of 2−4 days at 4 °C. The ZnPP UV−vis absorption spectrum was not affected by these manipulations, and no loss of porphyrin was detected. Photosensitized Generation of H2: Dimer/24-mer Dependence. Continuous white light irradiation of anaerobic solutions containing N148D ZnPP-Bfr, Pt NP@citrate, and ascorbate in MOPS buffer stimulated a steadily increasing accumulation of headspace H2 over the course of 6 h (Figure 3). The rate and yield of H2 were dependent on the relative proportions of dimer and 24-mer oligomers. At the same reagent and total ZnPP concentrations, irradiated solutions containing lower proportions of dimer and correspondingly higher proportions of 24-mer showed significantly slower accumulation and lower yields of H2. A solution containing 10% dimer and 90% 24-mer gave approximately one-third the 6 h yield of H2 as that containing ∼100% dimer (Figure 3, top panel). Pre- and postirradiation samples showed no change in the oligomer composition based on SEC. The dimeric form of N148D ZnPP-Bfr is thus much more active than the 24-mer in photosensitized H2 generation. The UV−vis absorption spectra of ZnPP in the N148D ZnPP-Bfr dimer and ZnPP wtBfr 24mer are superimposable in MOPS buffer,19 indicating no drastic differences in ZnPP environment. The propionate side of ZnPP is oriented toward the interior of the protein shell (Figure 1B), and conversion to the dimeric form may increase exposure of the ZnPP to electron donors or acceptors, thereby increasing the efficiency of photosensitized H2 generation. All further results described below used N148D ZnPP-Bfr stock solutions that had been characterized as ∼100% dimer by SEC and diluted to well below the concentrations required for conversion to 24-mer. The dimeric nature of the N148D ZnPP-Bfr used for the H2 generation in MOPS buffer was confirmed by DLS (which is shown together with other DLS results described below). Photosensitized Generation of H2. Dependence on the Various Components. Under our conditions with Pt NP@citrate (30 μM Pt and 2 mM ascorbate in 50 mM MOPS pH 7.4), photosensitized H2 generation was found to be optimal at 15 μM N148D ZnPP-Bfr dimer (Figure 3, bottom panel), reaching 5.7 ± 0.4 μmols H2, which corresponds to a turnover of 130 ± 9 H2/ZnPP at 6 h of continuous irradiation. Control solutions individually omitting Pt NPs, ascorbate, or N148D ZnPP-Bfr did not generate detectable H2 over at least fours hours of continuous white light irradiation. Unirradiated complete mixtures also did not generate detectable H2. The analogous system using Pt NP@pf-wtBfr in place of Pt NP@ citrate at the same Pt concentration also showed photosensitized H2 production but with 6 h yields (2.3 ± 0.3 μmols), 30−40% of that with Pt NP@citrate (Figure 4). Addition of 2

Figure 3. Top panel: Dependence of photosensitized H2 generation on N148D ZnPP-Bfr dimer/24-mer proportions. All solutions contained 15 μM total ZnPP in N148D ZnPP-Bfr and 2 mM sodium ascorbate in 50 mM MOPS pH 7.4. Listed oligomer percentages are on a protein basis (A280 nm), as determined by SEC (Figure S2). Bottom panel: Dependence of photosensitized H2 generation on concentrations of 100% dimeric N148D ZnPP-Bfr at 15 (closed circles), 30 (open circles), or 7 (closed squares) μM. “0” (open squares) indicates no protein present. All solutions contained 30 μM Pt as Pt NP@citrate and 2 mM ascorbate in 50 mM MOPS pH 7.4.

mM citrate to the optimized ascorbate/Pt NP@citrate/N148D ZnPP-Bfr dimer/MOPS mixture had no effect on photosensitized H2 time courses or yields. When 2 mM citrate was substituted in place of ascorbate, no photosensitized H2 production was observed over 4 h of irradiation. We visually observed no precipitation or cloudiness in any of these solutions during irradiations. Changing the MOPS concentration from 50 mM to either 100 mM or 25 mM in the otherwise optimized mixture (15 μM N148D ZnPP-Bfr dimer, 30 μM Pt as Pt NP@citrate, and 2 mM ascorbate) had no significant effect on photosensitized H2 production (Figure 5, top panel). However, decreasing the MOPS concentration to 5 mM resulted in much lower yields of H2 (0.8 ± 0.1 μmols), and H2 generation ceased after 3 h irradiation. An ∼25% loss of ZnPP Soret absorption was observed during 4 h irradiation of the complete mixture in 5 mM MOPS (Figure 5, bottom panel). In the absence of MOPS (at pH ∼7 with no other buffer or salt) in the otherwise complete optimized mixture, the Soret absorption intensity of N148D ZnPP-Bfr dimer decreased by ∼60% during 4 h of 4587

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Figure 4. Time dependence of photosensitized H2 generation in solutions containing 15 μM N148D ZnPP-Bfr dimer, 2 mM ascorbate, and 30 μM Pt as Pt NP@citrate (closed circles), 30 μM Pt as Pt NP@ pf-wtBfr (open circles), or no Pt (closed squares). All solutions were in 50 mM MOPS, pH 7.4.

irradiation, and no H2 was detected. These observations are consistent with MOPS functioning as SED and with ascorbate’s inability to function as an SED in our system. The 6 h yield of H2 (∼6 μmols) with 2 mM ascorbate is approximately the same as the initial amount of ascorbate present, which might seem consistent with ascorbate functioning as a sacrificial electron donor. However, increasing the initial ascorbate concentration to 50 mM resulted in no change in H2 generation rate or yield compared to 2 mM initial ascorbate. Lowering the concentration of ascorbate from 2 to 0.1 mM decreased the 6 h photosensitized H2 yield to 1.4 ± 0.3 μmols of H2, and H2 production ceased between 4 and 5 h irradiation (Figure 6, top panel). This lower H2 yield, however, is significantly higher than that of the initial ascorbate (0.3 μmols). These effects suggest another role for ascorbate. In fact, ascorbate appeared to exert a protective effect on ZnPP in N148D ZnPP-Bfr during irradiation in the presence of Pt NP@ citrate. At an initial ascorbate concentration of 2 mM, UV− visible absorption spectral time courses showed ∼15% loss of Soret absorption intensity during 6 h of white light irradiation (Figure 6, middle panel), whereas at 0.1 mM initial ascorbate a >50% loss of Soret absorption was observed over the same time period (Figure 6, bottom panel). We were able to monitor consumption of ascorbate at this lower concentration by loss of its 265 nm absorption27 (Figure 6, bottom panel). Fifty millimolar initial ascorbate did not stabilize the ZnPP Soret absorption to a greater extent than that at 2 mM. Although EDTA is among the most frequently used SEDs for photosensitized H2 production, we found that EDTA inhibited photosensitized H2 production by the optimized N148D ZnPPBfr dimer/Pt NP@citrate/ascorbate/MOPS system. The presence of 50 mM EDTA decreased the 4 h H2 yield by ∼50% (2.2 ± 0.2 μmols H2) (Figure 7). Equivalent solutions with 50 mM EDTA and no ascorbate generated even less H2 (0.5 ± 0.2 μmols) after 4 h irradiation. This latter observation could be related to the protective effect of ascorbate on the ZnPP described above. Substituting 50 mM HEPES, pH 7.4 or 50 mM MES, pH 6 in place of MOPS in the optimized Pt NP@citrate/N148D ZnPP-Bfr/ascorbate mixtures had little or no effect on

Figure 5. Top panel: Dependence of ZnPP-Bfr photosensitized H2 generation on MOPS concentration. All solutions contained 15 μM N148D ZnPP-Bfr, 30 μM Pt as Pt NP@citrate, and 2 mM ascorbate, and the millimolar MOPS concentrations of 100 (black), 50 (blue), 25 (red), and 5 (green) are all at pH 7.4. The “0” MOPS (purple) contained no other buffer or salt, and the pH was ∼7. Bottom panel: UV−vis absorption spectral changes in solutions containing 10 μM ZnPP-Bfr, 30 μM Pt as Pt NP@citrate, and 2 mM ascorbate in 5 mM MOPS, pH 7.4. UV−vis absorption spectra were obtained every 60 min during the 4 h irradiation period.

photosensitized H2 production rates or yields over 6 h irradiation compared to that in 50 mM MOPS (Figure 8). However, in analogous phosphate buffered solutions (no MOPS), only a very small amount of H2 was detectable and only after more than 3 h of continuous irradiation (Figure 8). Analogous solutions containing 50 mM NaCl in 50 mM MOPS, pH 7.4 also yielded no detectable H2 after 4 h irradiation. The N148D ZnPP-Bfr Soret band absorption was rapidly lost (within 1 to 2 h) during irradiation of the solutions containing NaCl in MOPS. DLS Sizing of Particles in Mixtures of N148D ZnPP-Bfr with Pt NPs. Because we observed photosensitized generation of H2 in the absence of an added electron transfer mediator, we used DLS to investigate the possibility of association between Pt NPs and N148D ZnPP-Bfr. Results are presented in Figure 9 and Table 1. DLS of solutions containing Pt NP@citrate and N148D ZnPP-Bfr dimer in 50 mM MOPS (Figure 9, purple trace) showed a particle with a mode diameter of 2.7 nm and a larger particle of mode diameter 4.1 nm. The diameter of the smaller particle is the same as that of the Pt NP@citrate. The larger particle could be due to association of Pt NP@citrate 4588

DOI: 10.1021/acs.inorgchem.7b00228 Inorg. Chem. 2017, 56, 4584−4593

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Figure 7. Time dependence of photosensitized H2 generation in solutions containing 50 mM EDTA and either 2 mM ascorbate (closed circles) or no ascorbate (open circles). All solutions contained 15 μM N148D ZnPP-Bfr dimer and 30 μM Pt as Pt NP@citrate in 50 mM MOPS, pH 7.4.

Figure 8. Photosensitized H2 generation in various buffers and pHs. Solutions contained 15 μM N148D ZnPP-Bfr, 30 μM Pt as Pt NP@ citrate, 2 mM ascorbate, and 50 mM of MOPS (closed circles), HEPES (closed squares), MES (open circles), 50 mM phosphate (open squares), or 50 MOPS + 50 mM NaCl (open triangles). All solutions were at pH 7.4 except MES, which was at pH 6.

aggregates may also contain Pt NP@citrate. DLS of N148 ZnPP-Bfr dimer + Pt NP@pf-wtBfr mixtures in MOPS showed no clear evidence for association between the two particles, and no larger aggregates were observed. (Figure 9, blue trace). The DLS traces of the same mixtures after irradiation were not significantly different from those shown in Figure 9. The relative intensities and peak areas within an individual DLS trace of these mixtures do not necessarily reflect relative amounts of each species. Pt NP@citrate, N148 ZnPP-Bfr dimer, and Pt NP@pf-wtBfr (24-mer) may have significantly differing inherent scattering properties. Comparison with Photosensitization by ZnTMPyP. Surprisingly, we detected no photosensitized H2 generation in an analogous 2 mM ascorbate/30 μM Pt as Pt NP@citrate/50 mM MOPS mixture when the water-soluble ZnTMPyP was substituted in place of N148D ZnPP-Bfr at the same porphyrin concentration (15 μM). Consistent with previous reports, we observed photosensitized H2 generation from ZnTMPyP/ MV2+/EDTA/Pt NP@citrate solutions in 50 mM MOPS (Figure S3), which presumably occurred via Scheme 1.14,15 We found that addition of ascorbate (2 mM) to this MV-containing

Figure 6. Comparisons of ascorbate concentrations on photosensitized H2 production (top panel) and UV−vis absorption time courses (bottom two panels). All solutions contained 30 μM Pt as Pt NP@ citrate and 15 μM N148D ZnPP-Bfr dimer in 50 mM MOPS pH 7.4 with either 2 or 0.1 mM initial sodium ascorbate, as indicated within the panels. UV−vis absorption spectra were obtained every 30−60 min during the 6 h irradiation.

with N148D ZnPP-Bfr dimer. In the absence of buffer (and no salt), DLS sizing of N148D ZnPP-Bfr dimer + Pt NP@citrate mixtures showed no change in NP diameters or dispersion compared to those in 50 mM MOPS. However, at higher ionic strengths mixtures of Pt NP@citrate and N148D, ZnPP-Bfr dimer showed aggregation into much larger particles. In 50 mM phosphate buffer or 50 mM NaCl in 50 mM MOPS, the DLS indicated particles near the diameter expected for a Bfr 24-mer (10−12 nm) but with greater size dispersion as well as larger aggregates (∼200 nm) (Figure 9, red and green traces). These 4589

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DISCUSSION The photosensitized generation of H2 in our N148D ZnPPBfr/Pt NP@citrate/ascorbate/MOPS system could conceivably occur via either oxidative or reductive quenching of 3ZnPP*, as diagrammed in Scheme 2. In the absence of an added electron Scheme 2. Possible Oxidative and Reductive Quenching Pathways for Photosensitized H2 Production by N148D ZnPP-Bfr Dimer and Pt NPs

relay molecule, these quenching pathways imply direct electron transfer from either 3ZnPP* or ZnPP•− within an N148D ZnPP-Bfr dimer to a Pt NP which, in our system, was coated with either citrate or pf-wtBfr. The photosensitized H2 yield at 5 mM MOPS was significantly lower than those at 25, 50, or 100 mM MOPS (Figure 5), and photosensitized H2 production was not observed in the absence of MOPS. We also found that photosensitized H2 generation was equally efficient in 50 mM of other Good’s buffers (HEPES, pH 7.4 or MES, pH 6) as in MOPS, whereas we observed little or no photosensitized H2 generation in phosphate buffer (Figure 8). These observations are consistent with an SED function for these Good’s buffers. Their structures are shown in Figure 10. Like other aliphatic

Figure 9. DLS traces of solutions containing 3 μM N148D ZnPP-Bfr and no Pt (black), ∼6 μM Pt as Pt NP@citrate in MOPS (purple), Pt NP@citrate in phosphate (red), Pt NP@citrate in MOPS + NaCl (green), or Pt NP@pf-wtBfr in MOPs (blue). All solutions were at pH 7.4 and, where present, MOPS, phosphate, or NaCl were all at 50 mM. Top and bottom panels show the same data in the 0−20 nm and 0− 300 nm ranges, respectively. In the top panel, the vertical line labeled “dimer” above the black trace indicates the position of the mode diameter for N148D ZnPP-Bfr dimer in MOPS with no other added components.

Table 1. Sizes of Pt NPs, N148D ZnPP-Bfr Dimer, and Their Mixtures Determined by DLS particle(s) a

Pt NP@pf-wtBfr Pt NP@citratea N148D ZnPP-Bfr dimerb N148D ZnPP-Bfr dimer + Pt NP@citrate/ MOPSb N148D ZnPP-Bfr dimer + Pt NP@citrate/ phosphateb N148D ZnPP-Bfr dimer + Pt NP@citrate/ MOPS/NaClb a

diameter range (nm)

mode diameter (nm)

7.5−11.7 2−4.9 1.7−4.2 2−8.7

10.1 2.7 2.3 4.1

1.7−220

10.7

2.5−300

11.7

Figure 10. Structures of Good’s buffers used in this work.23,24,29

tertiary amines, Good’s buffers can function as reducing agents30,31 and as SEDs in photosensitized reactions.32,33 In these roles, the tertiary amine is typically oxidized to an aminecentered cation radical. This initial radical can deprotonate to a much more reactive α-amino carbon-centered radical which undergoes a second one-electron oxidation followed by irreversible decomposition.34,35 Amine cation radicals have been detected during oxidations of piperazinyl Good’s buffers,36,37 and effects of MOPS and MES on radical reactions are consistent with analogous amine radical formation.38,39 In the relatively low ionic strength zwitterionic MOPS buffer, DLS showed the presence of a particle whose size is consistent with that for a complex between N148D ZnPP-Bfr dimer and Pt NP@citrate. In the same buffer, DLS did not detect a complex between N148D ZnPP-Bfr dimer and Pt NP@pfwtBfr. This latter pairing was also able to photosensitize H2 generation, albeit in ∼50% lower yield than with Pt NP@

From DLS traces in Figure 2. bFrom DLS traces in Figure 9.

system drastically inhibited photosensitized H2 production, which can be attributed to recycling of MV•+ by ascorbyl radical.28 In the absence of EDTA, no photosensitized H2 was produced in ZnTMPyP/MV2+/Pt NP@citrate/MOPS solutions containing 2 mM ascorbate. 4590

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proficient than MOPS at photoinduced overreduction of ZnPP in N148D Bfr dimer. This possibility is consistent with the much lower photosensitized H2 yield in our system in the presence of EDTA without ascorbate and with ascorbate’s mitigation of this lower yield (Figure 7). Reductive scavenging of SEDox radicals constitutes a heretofore undefined but not unreasonable role for ascorbate in porphyrin-based photosensitized H2 generation.

citrate. These observations indicate that formation of a longlived, discrete complex containing N148D ZnPP-Bfr dimer and Pt NP may facilitate but is not essential for photosensitized H2 generation in our system. Higher ionic strength (50 mM phosphate or 50 mM NaCl) caused aggregation into much larger particles and essentially shut down photosensitized H2 generation. The aggregation at higher ionic strength is consistent with the expected decreased surface charge repulsion among the particles40 and could restrict productive access among the various components. The lower ionic strength Good’s buffers thus minimized formation of unreactive large aggregates while still allowing productive associations of N148D ZnPP-Bfr dimer with Pt NP@citrate and Pt NP@pfwtBfr. Ascorbate has often been used as an SED with metalpolypyridyl PSs4,41 but only rarely with ZnP PSs, where ascorbate appears to be much less efficient than tertiary amines.13,42 We are unaware of any reports in which ascorbate functions as direct electron donor or electron relay to colloidal Pt for H2 generation. Ascorbate as an SED is typically used at acidic pHs (4−6) and on the order of 100 mM. At the relatively low ascorbate concentration (2 mM) and higher pH (7.4) used in our optimized system, ascorbate is unlikely to function as the primary SED. Indeed, the photosensitized H2 yield exceeded the two-electron donating capacity of 0.1 mM ascorbate in our system, and 50 mM ascorbate did not significantly increase photosensitized H2 yield over that with 2 mM ascorbate. In our system, ascorbate appeared to protect the ZnPP in Bfr from photodegradation, as shown in Figure 6. This protection likely reflects ascorbate’s well-established biological role as a radical scavenging antioxidant.27,43,44 Previous studies showed that with EDTA as the SED and the absence of an electron acceptor, the water-soluble ZnTMPyP underwent rapid photodegradation via reductive quenching of the 3ZnP* to ZnP•−, followed by dark reduction to the zinc dihydroporphyrin (phlorin) and further decomposition.15,45 Ascorbate (in its deprotonated form, AscH− at pH 7) may protect ZnPP•− in Bfr against analogous photoinduced overreduction by reductively scavenging the Good’s buffer amine cation radical resulting from sacrificial electron donation to 3ZnPP*, as diagrammed in Scheme 3 for MOPS. The resulting amine-centered cation

2Asc•− + H+ → AscH− + DHA

(1) 3

Both oxidative and quenching of ZnPP* in proteins has been observed.50 Due to the irreversibility of the various redox processes, measured or estimated thermodynamic redox potentials do not always provide a reliable basis for choosing between oxidative and reductive quenching pathways.4 For example, cyclic voltammetry showed only irreversible oxidation waves at ∼0.8 V vs NHE for various Good’s buffers30,36 and ∼1.5 V for EDTA.4 Our rationale for the protective role of ascorbate invokes reduction of 3ZnPP* to ZnPP•− by Good’s buffers (Scheme 3), implying that our photoinduced H2generating system operates via the reductive quenching pathway shown in Scheme 2. This conclusion is consistent with previous results on the EDTA/TMPyP/Pt NP system in the absence of MV.15,16 However, we cannot rule out the possibility that the reductive quenching in our system is a side reaction and that the photosensitized H2 generation proceeds via the oxidative quenching pathway. The inability of the analogous ascorbate/ZnTMPyP/Pt NP@citrate/MOPS system to photosensitize H2 generation could be due to inefficient reductive quenching of 3ZnTMPyP* by MOPS and/or irreversible photoinduced reduction of ZnTMPyP•−.45

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CONCLUSIONS We demonstrated the first example of a protein-bound ZnP as photosensitizer for H2 generation in aqueous solution without an added electron relay. Photosensitized H2 turnovers per ZnPP or per Pt in our ascorbate/ZnPP-Bfr/Pt NP@citrate system were at least triple those of our previously reported ZnPP-Bfr system19 and also exceeded those of other proteinbound ZnP systems.17,18 Unlike that reported here, these previous systems required a viologen as an electron relay molecule and operated according to Scheme 1. Bfr thus provides a versatile, readily modifiable scaffold for manipulation of porphyrin-type PSs in aqueous solution.

Scheme 3. Proposed Scavenging of MOPS•+ by AscH− during Reductive Quenching of 3ZnPP* in N148D ZnPP-Bfr Dimer

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00228. Sampling and GC calibration procedures, SEC of N148D ZnPP-Bfr, TEM of Pt NP@citrate, and ZnTMPyPphotosensitized H2 production with MV (PDF)

radical, MOPS•+, would be reduced back to MOPS by AscH−, and the ascorbyl radical (Asc•−) would then rapidly disproportionate to ascorbate (AscH−) and dehydroascorbate (DHA) (Reaction 1),27,46 thereby providing a second reductively scavenging AscH− (significant rereduction of DHA is unlikely under our conditions47−49). Scavenging of MOPS•+ by AscH− would minimize conversion to the more reactive deprotonated carbon-centered radical (MOPS•), which could irreversibly reduce ZnPP•−. The analogous amine or carbon-centered radicals of the sacrificial electron donor, EDTA, could be more

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Donald M. Kurtz Jr.: 0000-0003-1179-1875 Notes

The authors declare no competing financial interest. 4591

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ACKNOWLEDGMENTS This research was supported by a grant from the American Chemical Society Petroleum Research Fund (Grant 49201ND4 to D.M.K.). E.C. received financial support from a University of Texas Board of Regents Graduate Program Initiative grant. We thank Professor Heather Shipley in the UTSA Department of Civil and Environmental Engineering for access to and assistance with ICP-OES.

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