Research overview:

Research in the Milton Group seeks to couple metalloenzymes that catalyze important reductive reactions to electrodes, in order to study their electron transfer/catalytic mechanisms and ultimately aid the development of new biotechnologies (and bio-inspired technologies).

Our multidisciplinary research combines skills ranging from recombinant protein production to enzymatic electrochemistry to study enzymes that catalyze reactions such as (i) dinitrogen (N2) reduction to ammonia (NH3), (ii) carbon dioxide (CO2) reduction, and (iii) large metalloenzyme complexes that employ non-trivial electron transfer mechanisms. In enzymatic electrochemistry, electrodes can be electronically coupled to metalloenzymes in many different ways although the desired outcome remains the same: the electrode supplies the reducing equivalents to the reductive metalloenzyme for subsequent catalysis (or, electrocatalysis) where the corresponding current is proportional to the rate of substrate reduction by the enzyme. 




Nitrogenase Figure V2.png






As the only known metalloenzyme capable of reducing kinetically inert dinitrogen (to produce ammonia), nitrogenase is of significant research interest. First, understanding how nitrogenase transfers electrons and hydrolyzes ATP to reduce dinitrogen (in aqueous solution) could assist the design of bio-inspired catalysts for ammonia production. The decoupling of nitrogenase from its necessity to hydrolyze ATP represents an additional valuable target. In 2016, it was shown that an electrode could be used to bypass the electron-supplying, ATP-hydrolyzing Fe protein of nitrogenase and supply electrons to the catalytic MoFe protein for substrate reduction. To this end, one of the aims of our research group is it develop advanced nitrogenase-electrode interfaces to study this enzyme's mechanism, ultimately progressing towards the deployment of nitrogenase in new biotechnologies for ammonia production. Take a look at this review of our work.


Figure (top right). Representation of the nitrogenase complex. Each active dimer of the tetrameric catalytic MoFe protein contains an iron-molybdenum cofactor (FeMo-co, [7Fe-9S-C-Mo-homocitrate]) where dinitrogen and protons are reduced to two equivalents of ammonia and one equivalent of molecular hydrogen. A [8Fe-7S] P cluster within the MoFe protein is involved in transferring electrons from the reducing Fe protein, which contains a [4Fe-4S] cluster and two MgATP-hydrolyzing sites. (Bottom right). Structures of nitrogenase's cofactors, where the position colored in cyan can be a Mo, V, or Fe metal. Fe = rust, S = yellow, C = beige, Mo/V/Fe = cyan, O = red.











Dark-operative protochlorophyllide oxidoreductase (DPOR):DPOR.png

Chlorophylls and bacteriochlorophylls are organic pigments that are essential to light-harvesting organisms, produced in nature on a global scale of around 6 billion tons annually. DPOR is a complex metalloenzyme that catalyzes the light-independent 2-electron reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) in phototrophic bacteria, which undergoes a second 2-electron reduction to yield bacteriochlorophyllide (Bchlide). We are interested in understanding the catalytic and electron transfer mechanisms of this metalloenzyme complex, which shares a degree of homology with nitrogenase. DPOR also couples the hydrolysis of ATP to electron transfer for substrate reduction.

This research is funded by the Swiss National Science Foundation (SNSF), grant number: 200021_191985.


Figure (right). Representation of the DPOR complex. Each catalytic half of the tetrameric catalytic protein (here, BchNB) contains a [4Fe-4S] cluster that presumably aids in transferring electrons from the reducing (and MgATP-hydrolyzing) reductase "BchL", for Pchlide reduction within the BchNB protein. Being similar to the Fe protein of nitrogenase, the BchL protein contains a single [4Fe-4S] cluster in addition to two MgATP-hydrolyzing sites. Illustration prepared from Protein Data Bank file 2YNM.

Figure (below). DPOR catalyzes the 2-electron reduction of Pchlide to Chlide, at the C17=C18 double bond in the D ring of the substrate. In phototrophic bacteria, chlorophyllide oxidoreductase (COR) performs a second 2-electron reduction of Chlide to yield bacteriochlorophyllide (Bchlide).















Formate dehydrogenase (Fdh)Fdh and Hyd.png

Formate dehydrogenases are enzymes that catalyze the reduction of carbon dioxide to formate (or formate oxidation to carbon dioxide). Fdhs can be further divided into metal-dependent Fdhs (W- or Mo-dependent) and metal-independent Fdhs (such as NAD+-dependent). We are interested in metal-dependent Fdhs as they can undergo facile electron transfer with heterogeneous surfaces (such as electrode surfaces) and catalyze carbon dioxide reduction at high rates. To this end, we are interested in developing bespoke/tailored electrode:enzyme interfaces for enhanced electroenzymatic carbon dioxide reduction by Fdhs.

Figure (right): Representation of metal-dependent formate dehydrogenase. In this case, the Mo-dependent Fdh from Escherichia coli is shown (adapted from Protein Data Bank file 1AA6). This Fdh also contains a selenocysteine residue in close proximity to the molybdenum cofactor (Moco) where carbon dioxide is reduced.





Hydrogenases are metal-containing enzymes that reduce protons to produce molecular hydrogen (or vice versa). Thus, hydrogenases are attractive for new biotechnological and electrochemical approaches to either produce electrical energy from the oxidation of molecular hydrogen, or to produce molecular hydrogen from renewable electrical energy (as a form of energy storage). In our research group, we typically employ hydrogenase as a model hydrogen-evolving metalloenzyme when we are developing electron mediators and/or tailored electrode surfaces for enzymatic electrochemistry. While there are 4 main types of hydrogenases ([NiFe], [NiFeSe], [FeFe] and [Fe]-only), we employ the [FeFe]-hydrogenase from Clostridium pasteurianum.

Figure (right): Representation of [FeFe]-hydrogenase from C. pasteurianum. Adapted from Protein Data Bank file 1FEH. 







Financial support:









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