Light‐Driven Hydrogen Evolution by Nickel‐Substituted Rubredoxin

Abstract

An enzymatic system for light-driven hydrogen generation has been developed throughc ovalent attachment of ar uthenium chromophore to nickel-substituted rubredoxin (NiRd).The photoinduceda ctivity of the hybrid enzymei ss ignificantly greater than that of at wo-component system and is strongly dependent on the positiono ft he ruthenium phototriggerr elative to the active site, indicating ar ole for intramolecular electron transfer in catalysis. Steady-state and time-resolved emission spectra reveal ap athway for rapid, directq uenching of the ruthenium excited state by nickel, butl ow overall turnover numberssuggest initial electron transfer is not the rate-limiting step. This approach is ideally suited for detailed mechanistic investigationso fc atalysis by NiRd and other molecular systems, with implications for generation of solar fuels.Hydrogeni sc onsidered to be ac lean, renewable alternative to carbon-based fuels. However, generating hydrogen through traditional methods such as electrolytic water splitting can be costly and energy intensive, thus overriding the advantages of this supposedly sustainable fuel. [1][2][3] In contrast to anthropogenic approaches, biological systems have evolvedt op roduce hydrogen under mild conditions by using proteins called hydrogenases. [4,5] Although theseh ighly efficient enzymes suffer from extreme fragilityu nder atmospheric conditions, precluding general application, they have inspired the development of chemically diverse hydrogenase models. [6][7][8][9][10][11][12][13][14][15][16][17][18] Of particulari nterest is the design of systemst hat can be driven by light for the generation of so-called "solar fuels". [19][20][21][22][23][24][25] We have shown that nickel-substituted rubredoxin (NiRd),asmall electron-transfer protein that naturally binds iron in at etrahedral tetrathiolate coordination motif, mimics the structure and function of the redox-active site in the [NiFe] hydrogenases. The hydrogenevolvinga ctivity of this system has previously been characterized by using solution-based photochemical and electrocatalytic assays. [26] In this study,aruthenium chromophore was covalently attachedt oN iRd to directlyr educe the active site for light-initiated catalysis. Attachment at different locations aroundt he protein surface reveals as trong distance dependence for hydrogen evolution activity,a nd time-resolved emission studies were used to investigate the efficiency of electron transfer between the ruthenium and nickel centers. Low overall turnover numbers across all of the NiRd variants suggest initial electron transfer is not the rate-limiting step in catalysis.The ruthenium chromophore [Ru II (2,2'-bipyridine) 2 (5,6-epoxy-5,6-dihydro-[1,10]-phenanthroline)] 2 + was synthesized as previously reported and bound to the sulfhydryl group of af ree cysteiner esidue. [27] Following purification,t he labeling efficiency was found to be 90 AE 5% by optical absorption (see the Supporting Information, Figure S1). MALDI-TOF mass spectrometry confirmed this yield, showing ...

Going beyond Structure: Nickel-Substituted Rubredoxin as a Mechanistic Model for the [NiFe] Hydrogenases

“…28 Our group has expanded this characterization of NiRd activity using solution-phase, lightdriven, spectroscopic, and electrochemical measurements. 22,29,30 NiRd displays a moderate electrocatalytic overpotential of approximately 550 mV, which is similar to those seen for many first-generation small-molecule and recent protein-derived catalytic systems operating in purely aqueous solution, 31−38 and exhibits estimated electrocatalytic rates that are similar to those of the native enzymes. 22,30 As such, NiRd shows great potential as an artificial hydrogenase, as it is easily expressed, oxygen tolerant, chemically and thermally stable, and highly tunable for catalyst optimization.…”

“…22,29,30 NiRd displays a moderate electrocatalytic overpotential of approximately 550 mV, which is similar to those seen for many first-generation small-molecule and recent protein-derived catalytic systems operating in purely aqueous solution, 31−38 and exhibits estimated electrocatalytic rates that are similar to those of the native enzymes. 22,30 As such, NiRd shows great potential as an artificial hydrogenase, as it is easily expressed, oxygen tolerant, chemically and thermally stable, and highly tunable for catalyst optimization. 22 Further, the NiRd model system provides an opportunity to assess the specific contributions of the [Fe(CO)(CN) 2 ] fragment to activity in the native [NiFe] hydrogenases.…”

“…Additionally, the use of a structured protein provides a defined secondary coordination sphere, which is known to play an important role in modulating catalytic activity. − Previous work by Moura had shown that native rubredoxin proteins isolated from sulfate-reducing bacteria and substituted with nickel were active for hydrogen evolution and H/D exchange using traditional H 2 ase assays . Our group has expanded this characterization of NiRd activity using solution-phase, light-driven, spectroscopic, and electrochemical measurements. ,, NiRd displays a moderate electrocatalytic overpotential of approximately 550 mV, which is similar to those seen for many first-generation small-molecule and recent protein-derived catalytic systems operating in purely aqueous solution, − and exhibits estimated electrocatalytic rates that are similar to those of the native enzymes. , As such, NiRd shows great potential as an artificial hydrogenase, as it is easily expressed, oxygen tolerant, chemically and thermally stable, and highly tunable for catalyst optimization . Further, the NiRd model system provides an opportunity to assess the specific contributions of the [Fe­(CO)­(CN) 2 ] fragment to activity in the native [NiFe] hydrogenases. − …”

Biosynthetic Approaches towards the Design of Artificial Hydrogen‐Evolution Catalysts

“…Subsequently, photocatalytic H 2 evolution was studied (Figure ) by covalent attachment of a free Cys (Cys31) near the metal binding site using a ruthenium‐based chromophore [Ru II (2,2′‐bipyridine) 2 (5,6‐epoxy‐5,6‐dihydro‐[1,10]‐phenanthroline)] 2+ . The hybrid RuNiRd protein produced ≈110 nmol H 2 within 45 min corresponding to a TON of 3.5 and TOF of ≈0.1 min −1 .…”

Light‐driven catalysis with engineered enzymes and biomimetic systems

“…Although most hydrogenase mimicry has focused on the diiron hydrogenase, one of the earliest systems was based on the [NiFe] hydrogenase [155][156][157][158][159][160][161]. Substitution of Ni in rubredoxin yields a catalyst, Ni-Rd, with a tetrathiolate active site mimicking the primary coordination sphere of the Ni site in [NiFe] hydrogenase [155,161].…”