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The Armstrong Research Group
Inorganic Chemistry Laboratory

Third floor ICL, rooms: T7–T12, T17
Phone: T12 (Fraser’s office): +44 (0)1865 272647
e-mail: fraser.armstrong@chem.ox.ac.uk

Jump to : Carbon Monoxide Dehydrogenase  | Fumarate Reductase  | Nitrate Reductase  |

Carbon Monoxide Dehydrogenase

Carbon monoxide dehydrogenase (CODH) enzymes catalyse the interconversion of CO and CO2. These enzymes fall into two classes: Mo-CODH enzymes, found in aerobic carboxydotrophic bacteria which have a CuMo-pterin active site; and Ni-CODH enzymes, found in anaerobic bacteria which have a [Ni4Fe5S] cluster as the active site. There is also the related class of Ni-CODH/ACS, where the CODH enzyme is part of a larger protein in which CO2 reduction is coupled to acetyl CoA synthesis. These enzymes are remarkably efficient, and turnover rates of up to 40000 s-1 have been reported for CO oxidation by Ni-CODH at pH 8, 70 °C.

All of our work on CODH to date has focused on the enzymes 'CODH I' (137.0 kDa) and 'CODH II' (136.6 kDa) from the organism Carboxydothermus hydrogenoformans (Ch). These are two of five CODH enzymes expressed by this organism, and are closely related in structure. They are homodimers used in energy conservation (CODH I) and NADPH generation (CODH II), and are both of the Ni-CODH class. Crucially for us, both CODH I and CODH II adsorb electroactively onto pyrolytic graphite electrodes, which allows us to study them using protein film electrochemistry to monitor the effect of temperature, pH, determine Michaelis-Menten parameters, etc (Parkin et al., J. Am. Chem. Soc. 2007, 129, 10328-10329). More recently, we have exploited the great efficiency of CODH I in a graphite particle-based system in which we paired this enzyme with Hyd-2 from E. coli to create a catalyst for the water-gas shift reaction (conversion of CO and water into CO2 and hydrogen) (Lazarus et al., 2009, manuscript submitted for publication).

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Fumarate Reductase

Fumarate reductase (Frd) is an important respiratory enzyme, which catalyses the biological interconversion of fumarate and succinate. We have studied Frd extracted from Escherichia coli and Wolinella succinogenes. EcFRD and WoFRD are similar in structure; in the cell, they are membrane-bound and are found as tetramers: the four subunits are known collectively as FrdABCD. FrdABCD contains two quinone binding sites, three [Fe-S] clusters, which form an electron transfer chain from the enzyme's surface to the buried active site, which contains a covalently attached FAD (flavin adenine dinucleotide). In electrochemical experiments, the water-soluble sub-complex is used (FRDAB); this lacks the membrane anchors and the quinone binding sites but contains all three [Fe-S] clusters (found in FRDB) and the FAD group (FRDA)

We have studied the membrane-extrinsic catalytic domain, FrdAB, which contains the FAD and the [Fe-S] clusters, using protein film electrochemistry. Frd is damaged by O2 and so experiments on it have to be performed in an anaerobic glovebox, under an inert N2 atmosphere.The enzyme adsorbs to a pyrolytic graphite "edge" electrode to give characteristic voltammetric signals which can be resolved and assigned to the FAD and the three [Fe-S] clusters. Centre 2, [4Fe-4S], has a much lower equilibrium redox potential (-305 mV vs SHE) than the other centres, which have potentials around -50 mV vs SHE. Upon adding fumarate, the non-turnover signals undergo transformations as different centres become involved in catalytic turnover.

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Nitrate Reductase

Within the nitrogen cycle, the conversion of nitrate to nitrite is the central reductive process, and therefore studies of nitrate reduction are of great interest, with agricultural, environmental, and health implications. The nitrate reductase studied in this group is a respiratory enzyme located in the cytoplasmic membrane of the bacterium Escherichia coli. This nitrate reductase is a member of the DMSO reductase family of molybdoenzymes and catalyses the quinol-dependent reduction of nitrate to nitrite. In vivo, this enzyme is found as a membrane-bound complex of three subunits, known as NarG, NarH and NarI. NarG is the subunit of the enzyme which contains the catalytic centre and an [4Fe4S] cluster. NarH contains four iron-sulphur clusters; three [4Fe4S] clusters and one [3Fe4S] cluster. NarI is the integral membrane subunit and contains two b-type heme groups: the proximal 'bP' and the distal 'bD'.

E. coli nitrate reductase adsorbs to pyrolytic graphite and both wild-type and mutant forms have been investigated using protein film electrochemistry. Nitrate reductase research within the Armstrong group is conducted in collaboration with the group of Prof. Joel Weiner at the University of Alberta, Edmonton.

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