Fumarate reductase, voltammetry

Heering, H.A., Weiner, J.H., and Armstrong, F.A. (1997) Direct detection and measurement of electron relays in a multicentered enzyme voltammetry of electrode-surface films of E. coli fumarate reductase, an iron-sulfur flavoprotein. Journal of the American Chemical Society, 119,11628-11638. 

Unlike the E. coli fumarate reductase, flavocytochrome Cj (Fee,) from the marine bacterium Shew one lla frigidimarina is a soluble enzyme. It has four heme groups instead of Fe-S clusters, and the FAD is not covalently bound, but otherwise the active site and tire mechanism are similar to the E. coll enzyme. Conventional methods for studying the active site are not useful here, as the electronic spectrum of the flavin is masked by the intense transitions from the four heme groups, and the one-electron radical (which should be EPR active) is inherently unstable. As discussed above, the instability of the radical means that two electrons transfer cooperatively and this makes it easy to observe the FAD by voltammetry. ... 

In steady-state voltammetry experiments, enzyme activity is viewed in the potential domain that can pinpoint the role of centers as electron relays, or reveal the presence of internal control mechanisms, such as a redox transformation that causes the enzyme to switch ofF at a certain potential. Such studies can also reveal and quantify how an enzyme is redox-biased to favor catalysis in a particular direction. Figure 6(b) shows the voltammetry of a film of fumarate reductase obtained in the presence of a low concentration of fumarate and a high concentration of succinate, from which it is easily seen how the catalytic activity of the enzyme is biased heavily in the direction of fumarate reduction [38]. This experiment has been carried out with a rotating disc electrode. The current for succinate oxidation is independent of rotation rate, while that for fumarate reduction is very sensitive because the reaction is diffusion controlled. 

Fe 2S], a [4Fe-4S] and a [3Fe-4S] center. The enzyme catalyzes the reversible redox conversion of succinate to fumarate. Voltammetry of the enzyme on PGE electrodes in the presence of fumarate shows a catalytic wave for the reduction of fumarate to succinate (much more current than could be accounted for by the stoichiometric reduction of the protein active sites). Typical catalytic waves have a sigmoidal shape at a rotating disk electrode, but in the case of succinate dehydrogenase the catalytic wave shows a definite peak. This window of optimal potential for electrocatalysis seems to be a consequence of having multiple redox sites within the enzyme. Similar results were obtained with DMSO reductase, which contains a Mo-bis(pterin) active site and four [4Fe 4S] centers. 

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