Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal-Based Formate Dehydrogenase.

Metal-based formate dehydrogenases are molybdenum or tungsten-dependent enzymes that catalyze the interconversion between formate and CO2 . According to the current consensus, the metal ion of the catalytic center in its active form is coordinated by 6 S (or 5 S and 1 Se) atoms, leaving no free coordination sites to which formate could bind to the metal. Some authors have proposed that one of the active site ligands decoordinates during turnover to allow formate binding. Another proposal is that the oxidation of formate takes place in the second coordination sphere of the metal. Here, we have used electrochemical steady-state kinetics to elucidate the order of the steps in the catalytic cycle of two formate dehydrogenases. Our results strongly support the "second coordination sphere" hypothesis.

Electrochemical Studies of CO2 -Reducing Metalloenzymes.

Only two enzymes are capable of directly reducing CO2 : CO dehydrogenase, which produces CO at a [NiFe4 S4 ] active site, and formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy-efficient and specific in terms of product. They have been connected to electrodes with two different objectives. A series of studies used protein film electrochemistry to learn about different aspects of the mechanism of these enzymes (reactivity with substrates, inhibitors…). Another series focused on taking advantage of the catalytic performance of these enzymes to build biotechnological devices, from CO2 -reducing electrodes to full photochemical devices performing artificial photosynthesis. Here, we review all these works.

Formate Dehydrogenases Reduce CO2 Rather than HCO3- : An Electrochemical Demonstration.

Mo/W formate dehydrogenases catalyze the reversible reduction of CO2 species to formate. It is thought that the substrate is CO2 and not a hydrated species like HCO3- , but there is still no indisputable evidence for this, in spite of the extreme importance of the nature of the substrate for mechanistic studies. We devised a simple electrochemical method to definitively demonstrate that the substrate of formate dehydrogenases is indeed CO2 .

The two CO-dehydrogenases of Thermococcus sp. AM4.

Ni-containing CO-dehydrogenases (CODHs) allow some microorganisms to couple ATP synthesis to CO oxidation, or to use either CO or CO2 as a source of carbon. The recent detailed characterizations of some of them have evidenced a great diversity in terms of catalytic properties and resistance to O2. In an effort to increase the number of available CODHs, we have heterologously produced in Desulfovibrio fructosovorans, purified and characterized the two CooS-type CODHs (CooS1 and CooS2) from the hyperthermophilic archaeon Thermococcus sp. AM4 (Tc). We have also crystallized CooS2, which is coupled in vivo to a hydrogenase. CooS1 and CooS2 are homodimers, and harbour five metalloclusters: two [Ni4Fe-4S] C clusters, two [4Fe-4S] B clusters and one interfacial [4Fe-4S] D cluster. We show that both are dependent on a maturase, CooC1 or CooC2, which is interchangeable. The homologous protein CooC3 does not allow Ni insertion in either CooS. The two CODHs from Tc have similar properties: they can both oxidize and produce CO. The Michaelis constants (Km) are in the microM range for CO and in the mM range (CODH 1) or above (CODH 2) for CO2. Product inhibition is observed only for CO2 reduction, consistent with CO2 binding being much weaker than CO binding. The two enzymes are rather O2 sensitive (similarly to CODH II from Carboxydothermus hydrogenoformans), and react more slowly with O2 than any other CODH for which these data are available.

Studying direct electron transfer by site-directed immobilization of cellobiose dehydrogenase.

Covalent coupling between a surface exposed cysteine residue and maleimide groups was used to immobilize variants of Myriococcum thermophilum cellobiose dehydrogenase (MtCDH) at multiwall carbon nanotube electrodes. By introducing individual cysteine residues at particular places on the surface of the flavodehydrogenase domain of the flavocytochrome we are able to immobilize the different variants in different orientations. Our results show that direct electron transfer (DET) occurs exclusively through the haem b cofactor and that the redox potential of the haem is unaffected by the orientation of the enzyme. Electron transfer between the haem and the electrode is fast in all cases and at high glucose concentrations the catalytic currents are limited by the rate of inter-domain electron transfer (IET) between the FAD and the haem. Using ferrocene carboxylic acid as a mediator we find that the total amount of immobilized enzyme is 4 to 5 times greater than the amount of enzyme that participates in DET. The role of IET in the overall DET catalysed oxidation was also demonstrated by the effects of changing Ca2+ concentration and by proteolytic cleavage of the cytochrome domain on the DET and MET currents.

The effect of temperature on electrochemically driven denaturation monitored by SERS.

Scanning the electrochemical potential negative results in the gradual denaturation of dsDNA immobilised at a nanostructure gold electrode, the DNA melting is monitored by SERS. We demonstrate the effect of the experimental temperature on the electrochemically driven melting (E-melting) by carrying out experiments between 10 and 28 °C using two DNA duplexes (20 and 21 base pairs). Significant temperature dependence for both the melting potentials, Em, and the steepness of the melting curves was found over the range 10 to 18 °C. Above 18 °C the results were found to be independent of temperature. The relative temperature insensitivity of the melting potentials above 18 °C is advantageous for the application of the electrochemically driven melting technique because precise temperature control is not necessary for measurements that are carried out around room temperature. Conversely temperature dependence below 18 °C offers a way to improve discrimination for highly similar DNA sequences.