Publications by authors named "Ross D Milton"

Supramolecular surfactants provide a versatile platform to construct systems for solar fuel synthesis, for example via the self-assembly of amphiphilic photosensitizers and catalysts into diverse supramolecular structures. However, the utilization of amphiphilic photosensitizers in solar fuel production has predominantly focused on yielding gaseous products, such as molecular hydrogen (H), carbon monoxide (CO), and methane (CH) with turnover numbers (TONs) of synthetic catalysts typically in the range of hundreds to thousands. Inspired by biological lipid-protein interactions, we present herein a bio-hybrid assembly strategy that utilizes photosensitizers as surfactants to form micellar scaffolds that interface with enzymes, namely hydrogenases and formate dehydrogenases, for semi-artificial photosynthesis.

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The metalloenzyme [FeFe]-hydrogenase is of interest to future biotechnologies targeting the production of "green" hydrogen (H). We recently developed a simple two-step functionalized procedure to immobilize the [FeFe]-hydrogenase from Clostridium pasteurianum ("CpI") on mesoporous indium tin oxide (ITO) electrodes to achieve elevated H production with high operational stability and current densities of 8 mA cm. Here, we use a combination of atomic force microscopy (AFM), scanning electron microscopy (SEM) and electrochemical quartz crystal microbalance (EQCM) to understand how mesoporous ITO stabilizes and activates CpI for electroenzymatic H production.

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Nitrogenases catalyze dinitrogen (N) fixation to ammonia (NH). While these enzymes are highly sensitive to deactivation by molecular oxygen (O) they can be produced by obligate aerobes for diazotrophy, necessitating a mechanism by which nitrogenase can be protected from deactivation. In the bacterium Azotobacter vinelandii, one mode of such protection involves an O-responsive ferredoxin-type protein ("Shethna protein II", or "FeSII") which is thought to bind with Mo-dependent nitrogenase's two component proteins (NifH and NifDK) to form a catalytically stalled yet O-tolerant tripartite protein complex.

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The nitrogenase reductase NifH catalyses ATP-dependent electron delivery to the Mo-nitrogenase, a reaction central to biological dinitrogen (N) fixation. While NifHs have been extensively studied in bacteria, structural information about their archaeal counterparts is limited. Archaeal NifHs are considered more ancient, particularly those from Methanococcales, a group of marine hydrogenotrophic methanogens, which includes diazotrophs growing at temperatures near 92 °C.

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The assembly of semiconductors as light absorbers and enzymes as redox catalysts offers a promising approach for sustainable chemical synthesis driven by light. However, achieving the rational design of such semi-artificial systems requires a comprehensive understanding of the abiotic-biotic interface, which poses significant challenges. In this study, we demonstrate an electrostatic interaction strategy to interface negatively charged cyanamide modified graphitic carbon nitride (CN) with an [FeFe]-hydrogenase possessing a positive surface charge around the distal FeS cluster responsible for electron uptake into the enzyme.

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Modern societies rely heavily on centralized industrial processes to generate a multitude of products ranging from electrical energy to synthetic chemical building blocks to construction materials. To date, these processes have relied extensively on energy produced from fossil fuels, which has led to dramatically increased quantities of greenhouse gases (including carbon dioxide) being released into the atmosphere; the effects of the ensuing change to our climate are easily observed in day-to-day life. Some of the reactions catalyzed by these industrial processes can be catalyzed in nature by metal-containing enzymes (metalloenzymes) that have evolved over the course of up to 3.

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The biological N-fixation process is catalyzed exclusively by metallocofactor-containing nitrogenases. Structural and spectroscopic studies highlighted the presence of an additional mononuclear metal-binding (MMB) site, which can coordinate Fe in addition to the two metallocofactors required for the reaction. This MMB site is located 15-Å from the active site, at the interface of two NifK subunits.

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Massive efforts are invested in developing innovative CO -sequestration strategies to counter climate change and transform CO into higher-value products. CO -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction.

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The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as αß(γ) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases , previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.

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A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530).

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Enzymatic electrocatalysis holds promise for new biotechnological approaches to produce chemical commodities such as molecular hydrogen (H). However, typical inhibitory limitations include low stability and/or low electrocatalytic currents (low product yields). Here we report a facile single-step electrode preparation procedure using indium-tin oxide nanoparticles on carbon electrodes.

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The coupling of enzymes and/or intact bacteria with electrodes has been vastly investigated due to the wide range of existing applications. These span from biomedical and biosensing to energy production purposes and bioelectrosynthesis, whether for theoretical research or pure applied industrial processes. Both enzymes and bacteria offer a potential biotechnological alternative to noble/rare metal-dependent catalytic processes.

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Myoglobin (Mb) can react with hydrogen peroxide (H O ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo-peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo-Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands.

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Gas-processing metalloenzymes are of interest to future bio- and bioinspired technologies. Of particular importance are hydrogenases and nitrogenases, which both produce molecular hydrogen (H ) from proton (H ) reduction. Herein, we report on the use of rotating ring-disk electrochemistry (RRDE) and mass spectrometry (MS) to follow the production of H and isotopes produced from deuteron (D ) reduction (HD and D ) using the [FeFe]-hydrogenase from Clostridium pasteurianum, a model hydrogen-evolving metalloenzyme.

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Molecular hydrogen is a major high-energy carrier for future energy technologies, if produced from renewable electrical energy. Hydrogenase enzymes offer a pathway for bioelectrochemically producing hydrogen that is advantageous over traditional platforms for hydrogen production because of low overpotentials and ambient operating temperature and pressure. However, electron delivery from the electrode surface to the enzyme's active site is often rate-limiting.

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The fixation of atmospheric dinitrogen to ammonia by industrial technologies (such as the Haber Bosch process) has revolutionized humankind. In contrast to industrial technologies, a single enzyme is known for its ability to reduce or "fix" dinitrogen: nitrogenase. Nitrogenase is a complex oxidoreductase enzymatic system that includes a catalytic protein (where dinitrogen is reduced) and an electron-transferring reductase protein (termed the Fe protein) that delivers the electrons necessary for dinitrogen fixation.

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Hydrogenotrophic methanogens oxidize molecular hydrogen to reduce carbon dioxide to methane. In methanogens without cytochromes, the initial endergonic reduction of CO to formylmethanofuran with H-derived electrons is coupled to the exergonic reduction of a heterodisulfide of coenzymes B and M by flavin-based electron bifurcation (FBEB). In Methanococcus maripaludis, FBEB is performed by a heterodisulfide reductase (Hdr) enzyme complex that involves hydrogenase (Vhu), although formate dehydrogenase (Fdh) has been proposed as an alternative to Vhu.

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Increasing greenhouse gas emissions have resulted in greater motivation to find novel carbon dioxide (CO ) reduction technologies, where the reduction of CO to valuable chemical commodities is desirable. Molybdenum-dependent formate dehydrogenase (Mo-FDH) from Escherichia coli is a metalloenzyme that is able to interconvert formate and CO . We describe a low-potential redox polymer, synthesized by a facile method, that contains cobaltocene (grafted to poly(allylamine), Cc-PAA) to simultaneously mediate electrons to Mo-FDH and immobilize Mo-FDH at the surface of a carbon electrode.

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Over the past decade, there has been significant research in electrochemical reduction of CO, but it has been difficult to develop catalysts capable of C-C bond formation. Here, we report bioelectrocatalysis of vanadium nitrogenase from Azotobacter vinelandii, where cobaltocenium derivatives transfer electrons to the catalytic VFe protein, independent of ATP-hydrolysis. In this bioelectrochemical system, CO is reduced to ethylene (CH) and propene (CH), by a single metalloenzyme.

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Electrosynthesis of formate is a promising technology to convert CO and electricity from renewable sources into a biocompatible, soluble, non-flammable, and easily storable compound. In the model methanogen Methanococcus maripaludis, uptake of cathodic electrons was shown to proceed indirectly via formation of formate or H by undefined, cell-derived enzymes. Here, we identified that the multi-enzyme heterodisulfide reductase supercomplex (Hdr-SC) of M.

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Nitrogenase catalyzes the reduction of dinitrogen (N) to two ammonia (NH) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps.

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Enzymatic bioelectrocatalysis is being increasingly exploited to better understand oxidoreductase enzymes, to develop minimalistic yet specific biosensor platforms, and to develop alternative energy conversion devices and bioelectrosynthetic devices for the production of energy and/or important chemical commodities. In some cases, these enzymes are able to electronically communicate with an appropriately designed electrode surface without the requirement of an electron mediator to shuttle electrons between the enzyme and electrode. This phenomenon has been termed direct electron transfer or direct bioelectrocatalysis.

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Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD. We demonstrate how bioelectrocatalytic NAD/NADH inter-conversion can transform a glucose/O enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

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Nitrogenase, the only enzyme known to be able to reduce dinitrogen (N) to ammonia (NH), is irreversibly damaged upon exposure to molecular oxygen (O). Several microbes, however, are able to grow aerobically and diazotrophically (fixing N to grow) while containing functional nitrogenase. The obligate aerobic diazotroph, Azotobacter vinelandii, employs a multitude of protective mechanisms to preserve nitrogenase activity, including a "conformational switch" protein (FeSII, or "Shethna") that reversibly locks nitrogenase into a multicomponent protective complex upon exposure to low concentrations of O.

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As an essential component of amino acids and nucleic acids, nitrogen (N) is a key element of life. For atmospheric (dinitrogen, N ) and environmental (nitrate and nitrite, NO and NO ) sources of N to be utilized in amino acid synthesis in various forms of life, it must first be reduced to ammonia (NH ). The Haber-Bosch process, in which N is reduced to NH at elevated temperature and pressure, represents a major NH production process that has had a great impact on the agricultural crop industry.

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