GLOBAL OBSERVATION OF HYDROGEN DEUTERIUM ISOTOPE EFFECTS ON BIDIRECTIONAL CATALYTIC ELECTRON-TRANSPORT IN AN ENZYME - DIRECT MEASUREMENT BYPROTEIN-FILM VOLTAMMETRY/

Protein-Film voltammetry reveals the global effects of H/D isotopic su bstitutions, both in organic substrates and solvent, on bidirectional catalytic electron transport in a mitochondrial respiratory enzyme, su ccinate dehydrogenase. The voltammetry is controlled by the enzyme kin etics and therefore provides a direct display of the characteristic re lationships between turnover rates (current) and driving force (potent ial). This enables simultaneous measurement of relative electron-trans port rates for oxidative and reductive directions alongside thermodyna mic data (reduction potentials) relating to substrates and active site s. Measured over a range of pH and pD, the relationships between effec tive catalytic potentials of the enzyme and formal reduction potential s for substrate and active-site yield a ''potential-domain'' descripti on of the catalytic energetics, which complements and extends the pict ure obtained by conventional kinetic methods. For the organic substrat es, a marked decrease is observed in the rate of oxidation of perdeute riosuccinate compared to-succinate, but there is no change upon deuter ating fumarate. Can changing the solvent from H2O to D2O there is a si gnificant decrease in catalytic activity, particularly in the directio n of fumarate reduction. Simultaneously, the characteristic potentials of the enzyme (active-site FAD and effective catalytic potentials) ar e raised but to a lesser extent than is the reduction potential of the substrate. The catalytic energetics are thus altered. For the first t ime with a complex redox enzyme, the results enable H/D substitution e ffects to be rationalized in terms of the changes introduced in overal l driving force and enzyme reduction potentials, as well as the effect s of intrinsic kinetic processes.