ELECTROCATALYTIC VOLTAMMETRY OF SUCCINATE-DEHYDROGENASE - DIRECT QUANTIFICATION OF THE CATALYTIC PROPERTIES OF A COMPLEX ELECTRON-TRANSPORTENZYME

Succinate dehydrogenase (SDH), the membrane-extrinsic component of Com plex II, adsorbs at a pyrolytic graphite edge electrode and catalyzes interconversion of succinate and fumarate depending on the electrochem ical potential that is applied. The catalytic activity is measured ove r a continuous potential range, leading to a quantitative description of the interlinked energetics and kinetics of catalyzed electron trans port, including the degree to which. the enzyme is intrinsically tuned , at a particular pH, to function either in the direction of succinate oxidation or fumarate reduction. It is revealed that under reversible conditions (i.e. near the reduction potential of the fumarate/ succin ate couple) and at the physiological temperature of 38 degrees C, SDH is biased to catalyze fumarate reduction (reversal of the tricarboxyli c acid cycle) at pH values below 7.7. Subtle effects which gate electr on transport are detected. First, the sharp drop in catalytic activity observed as the potential is made more negative is an intrinsic prope rty that is associated with two-electron/two-proton reduction of the F AD, and second, binding and release of the competitive inhibitor/regul ator oxalacetate is observed as the enzyme is cycled between FAD(ox) ( tight binding) and FAD(red) (weaker binding) states. It is thereby dem onstrated how the electron-transport characteristics of a complex redo x enzyme, integrating both kinetic and thermodynamic information, can be derived from voltammetric experiments.