Charge Separation Related to Photocatalytic H2 Production from a Ru−Apoflavodoxin−Ni Biohybrid Sarah R. Soltau,†,§ Jens Niklas,† Peter D. Dahlberg,†,‡,∥ Karen L. Mulfort,† Oleg G. Poluektov,† and Lisa M. Utschig*,† †
Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, Illinois 60439, United States Graduate Program in Biophysics, The University of Chicago, Chicago, Illinois 60637, United States
‡
S Supporting Information *
ABSTRACT: The direct creation of a fuel from sunlight and water via photochemical energy conversion provides a sustainable method for producing a clean source of energy. Here we report the preparation of a solar fuel biohybrid that embeds a nickel diphosphine hydrogen evolution catalyst into the cofactor binding pocket of the electron shuttle protein, flavodoxin (Fld). The system is made photocatalytic by linking a cysteine residue in Fld to a ruthenium photosensitizer. Importantly, the protein environment enables the otherwise insoluble Ni catalyst to perform photocatalysis in aqueous solution over a pH range of 3.5−12.0, with optimal turnover frequency 410 ± 30 h−1 and turnover number 620 ± 80 mol H2/mol hybrid observed at pH 6.2. For the first time, a reversible light-induced charge-separated state involving a Ni(I) intermediate was directly monitored by electron paramagnetic resonance spectroscopy. Transient optical measurements reflect two conformational states, with a Ni(I) state formed in ∼1.6 or ∼185 μs that persists for several milliseconds as a long-lived charge-separated state facilitated by the protein matrix.
S
H−H bond formation. Studied extensively as electrocatalysts, there are comparatively few reported photocatalytic systems for water reduction utilizing these Ni-based catalysts. A notable example showcases the remarkable durability of a molecular [Ni(P2RN2R′)2]2+ catalyst in a homogeneous solar-driven catalytic H2 system, suggesting the potential for success in other photocatalytic systems.10 However, a significant concern for the applicability of these catalysts is the reported necessity of strongly acidic conditions for high rates of H2 formation in electrocatalytic studies.11−13 We were able to overcome this issue by self-assembling the molecular [Ni(P2PhN2Ph)2](BF4)2 catalyst (where Ph = phenyl) with the reaction center protein Photosystem I (PSI) and observed rapid photocatalysis at a near-neutral pH of 6.3.14 The large size and multiple cofactors of PSI, however, limited the ability to directly examine the lightdriven reactions related to H2 generation. In this Letter, we use the small protein flavodoxin (Fld) as a scaffold to create a photocatalytic hybrid, abbreviated Ru-ApoFld-Ni, for monitoring the effect of pH on light-driven catalysis and reduction of Ni(II). Importantly, this work exemplifies a useful protein-
ystems that use sunlight for the direct synthesis of energy-rich molecules, i.e. solar fuels, provide a valuable means of addressing global energy needs by providing renewable alternatives to fossil fuels.1,2 Of recent interest is the development of homogeneous synthetic and biohybrid architectures for the photocatalytic production of hydrogen, a clean and carbon-neutral fuel source.3−5 Further improvement of these systems relies on understanding essential underlying mechanisms for coupling photon capture to hydrogen generation. For this reason, we are targeting the creation of a new set of biohybrids that use small soluble proteins as scaffolds for directed binding of both light-harvesting and catalyst molecules.6,7 Importantly, these architectures enable the spectroscopic delineation of dynamic light-induced electron-transfer processes crucial to solar-driven proton reduction. The DuBois-type nickel-diphosphine molecular catalysts of the general form [Ni(P2RN2R′)2]2+ (where P2RN2R′ is 1,5-R′3,7-R-1,5-diaza-3,7-diphosphacyclooctane and R and R′ are substitutents on the phosphine and amine units, respectively) have emerged as some of the most active 3d transition-metal electrocatalysts for proton reduction.8,9 Key design features incorporated into the secondary coordination sphere of ligand structure are the pendant amines that function as proton relays, positioning the substrate near the active site and accelerating © XXXX American Chemical Society
Received: November 17, 2016 Accepted: December 19, 2016
230
DOI: 10.1021/acsenergylett.6b00614 ACS Energy Lett. 2017, 2, 230−237
Letter
http://pubs.acs.org/journal/aelccp
Letter
ACS Energy Letters
by loss of the MLCT band at ∼500 nm in the ultraviolet− visible (UV−vis) spectrum within minutes (Figure 2) and the
based strategy for stabilizing water insoluble catalysts in an aqueous regime for mechanistic studies of solar fuel generation. Flavodoxins are small (17 kDa), soluble electron carrier proteins that shuttle reducing equivalents between important cellular metabolic pathways. Synechococcus lividus Fld contains one cofactor, a flavin mononucleotide (FMN), which sits within a protein pocket but is not covalently attached to any Fld residues. FMN was removed from Fld using trichloroacetic acid precipitation,15,16 and [Ni(P2PhN2Ph)2](BF4)2 (NiC) (Figure 1B) was reconstituted with the resultant ApoFld at
Figure 2. UV−visible absorption spectra of S. lividus flavodoxin (black), ApoFld-Ni (red), [Ni(P2PhN2Ph)2](BF4)2 (NiC) in DMSO (blue), and NiC in buffer (green). Samples of protein were 11 μM in 20 mM Hepes pH 7.8, while samples of NiC were 90 μM NiC in DMSO and 60 μM NiC in 20 mM Hepes pH 7.8, with the spectrum recorded ∼1 min after dilution in the buffer from a 2 mM NiC stock in DMSO. Inset: protein samples of (A) native Fld, (B) ApoFld, and (C) ApoFld-Ni.
formation of a white precipitate (presumably the organic ligand, suggesting degradation of the metal complex). However, incorporation of NiC within ApoFld results in a highly stable complex, remaining active after storage for weeks at 4 °C, at least 6 months if stored at −80 °C, and through multiple freeze−thaw cycles. The NiC likely interacts through hydrophobic interactions with aromatic residues in the FMN binding pocket, specifically the indole ring of Trp 57 and the phenyl ring of Tyr 94,15,19 which stabilize a portion of the highly hydrophobic catalyst and maintain the structure of the FMN binding pocket (Figure 1C). Upon illumination, the Ru-ApoFld-Ni hybrid generates H2 from aqueous protons. We assert that the Fld structure facilitates H2 production by both solubizing the catalyst and positioning the catalyst in close proximity to RuPS (Figure 1C). On the basis of the crystal structure of S. elongatus of Fld (1CZL),18 the distance of a direct electron-transfer pathway is 10.2 Å between the RuPS binding site, Cys 54 (thiol), and the N3 of the FMN binding pocket. Photocatalysis experiments were performed using 1−2 μM protein, 100 mM sodium ascorbate, and 10 mM buffers ranging in pH from 3.5 to 12. H2 production was measurable across this entire pH range, with the best rate observed at pH 6.2 in 10 mM MES buffer with a turnover frequency (TOF) of 410 ± 30 h−1 and turnover number (TON) of 620 ± 80 mol H2/mol hybrid (Table 1, Figure S2). The quantum efficiency for the two-electron process of H2 production at pH 6.2 was determined to be 0.5 ± 0.1%. Photocatalysis without protein (solution of 2 μM NiC, 2 μM RuPS, and 100 mM sodium ascorbate) yielded low amounts of H2 (TOF 10 h−1 and TON 28 in 10 mM MES pH 6.2). To put this in context, our PSI system is the only other reported protein-based hybrid that incorporates a Ni diphosphine catalyst.14 In 10 mM MES buffer pH 6.3, very fast initial rates of H2 formation (TOF 2600 and 4500 h−1) and respectable TONs (1870 and 2824) were observed for NiC bound directly to PSI and NiC delivered to PSI via ApoFld, respectively. The >6-fold faster rates of PSI-driven versus
Figure 1. (A) Chemical structures of the molecular catalyst RuPS ([Ru(4-CH2Br-4′-CH3-2,2′-bpy) (bpy)2]·2PF6 and (B) NiC ([Ni(P2PhN2Ph)2](BF4)2) used in the current study. (C) Schematic representation of the Ru-ApoFld-Ni biohybrid using as a model the crystal structure of S. elongatus Fld (1CZL)18 superimposed with geometry-optimized models of the resting state catalyst and photosensitizer. A photocatalytic hydrogen production scheme is proposed, indicating electron transfer from the RuPS to NiC bound in the FMN binding pocket of ApoFld. Trp 57 and Tyr 94 are indicated in orange to designate potential hydrophobic interactions with NiC. Cys 54 is indicated in orange to show the covalent linkage between the Ru PS and the protein.
dilute concentrations during refolding of the protein, as has been previously described.14 Addition of excess native FMN cofactor to the ApoFld-Ni hybrid displaces Ni, confirming that at least a portion of the NiC molecule is noncovalently bound within the native flavin cofactor pocket provided by Fld (Figure S1). A Ru photosensitizer [Ru(4-CH2Br-4′-bpy) (bpy)2]·2PF6 (RuPS, where bpy = 2,2′-bipyridine) (Figure 1A) was covalently bound to the Cys 54 residue of ApoFld-Ni following overnight incubation. Specificity of RuPS binding was confirmed by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) modification17 of Cys 54 (1.0 Cys/ApoFld-Ni) preventing subsequent binding of RuPS to the protein (
