The use of biological systems for electrochemical energy conversion applications is often limited by instability of the protein or protein–electrode system. Here, we present a simple but efficient method for covalent attachment of nickel-substituted rubredoxin (NiRd), a model hydrogenase, to an unmodified graphite electrode based on amide bond formation. The resultant electrodes are shown to be highly active for H2 evolution over a period of several weeks. The effects of different attachment methods on interfacial electron transfer (ET) rates and catalysis are investigated, with decreased ET rates and increased background reactivity observed for surface-modified electrodes. Electrochemical simulations reveal that reduced protein dynamics of the attached NiRd enzyme are likely responsible for decreased catalytic rates by modulating the intramolecular proton transfer step. Ultimately, this straightforward approach can be broadly applied to diverse redox-active proteins and enzymes and will expand the utility of such systems by conferring increased stability over extended periods of time.

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The genetic encoding of artificial enzymes represents a substantial advantage relative to traditional molecular catalyst optimization, as laboratory-based directed evolution coupled with high-throughput screening methods can provide rapid development and functional characterization of enzyme libraries. However, these techniques have been of limited utility in the field of artificial metalloenzymes due to the need for in vitro cofactor metalation. Here, we report the development of methodology for in vivo production of nickel-substituted rubredoxin, an artificial metalloenzyme that is a structural, functional, and mechanistic mimic of the [NiFe] hydrogenases. Direct voltammetry on cell lysate establishes precedent for the development of an electrochemical screen. This technique will be broadly applicable to the in vivo generation of artificial metalloenzymes that require a non-native metal cofactor, offering a route for rapid enzyme optimization and setting the stage for integration of artificial metalloenzymes into biochemical pathways within diverse hosts.

Treatment of a dicopper(I,I) complex with excess amounts of NO leads to the formation of a dicopper dinitrosyl [Cu 2 (NO) 2 ] 2+ complex capable of (i) releasing two equivalents of NO reversibly in 90% yield and (ii) reacting with another equivalent of NO to afford N 2 O and dicopper nitrosyl oxo species [Cu 2 (NO)(O)] 2+ . Resonance Raman characterization of the [Cu 2 (NO) 2 ] 2+ complex shows a 15 N-sensitive N�O stretch at 1527.6 cm −1 and two Cu−N stretches at 390.6 and 414.1 cm −1 , supporting a symmetric diamond-core structure with bis-μ-NO ligands. The conversion of [Cu 2 (NO) 2 ] 2+ to [Cu 2 (NO)O] 2+ occurs via a rate-limiting reaction with NO and bypasses the dicopper oxo intermediate, a mechanism distinct from that of diFe-mediated NO reduction to N 2 O.

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