REVERSIBLE ELECTROCHEMISTRY OF FUMARATE REDUCTASE IMMOBILIZED ON AN ELECTRODE SURFACE - DIRECT VOLTAMMETRIC OBSERVATIONS OF REDOX CENTERS AND THEIR PARTICIPATION IN RAPID CATALYTIC ELECTRON-TRANSPORT

Fumarate reductase (Escherichia coli) can be immobilized in an extreme ly electroactive state at an electrode, with retention of native catal ytic properties. The membrane-extrinsic FrdAB component adsorbs to mon olayer coverage at edge-oriented pyrolytic graphite and catalyzes redu ction of fumarate or oxidation of succinate, depending upon the electr ode potential. In the absence of substrates, reversible redox transfor mations of centers in the enzyme are observed by cyclic voltammetry. T he major component of the voltammograms is a pair of narrow reduction and oxidation signals corresponding to a pH-sensitive couple with form al reduction potential E-degrees' = -48 mV vs SHE at pH 7.0 (25-degree s-C). This is assigned to two-electron reduction and oxidation of the active-site FAD. A redox couple with E-degrees' = -311 mV at pH 7 is a ssigned to center 2 ([4Fe-4S]2+/1+). Voltammograms for fumarate reduct ion at 25-degrees-C, measured with a rotating-disk electrode, show hig h catalytic activity without the low-potential switch-off that is obse rved for the related enzyme succinate dehydrogenase. The catalytic ele ctrochemistry is interpreted in terms of a basic model incorporating m ass transport of substrate, interfacial electron transfer, and intrins ic kinetic properties of the enzyme, each of these becoming a rate-det ermining factor under certain conditions. Electrochemical reversibilit y is approached under conditions of low turnover rate, for example, as thc supply of substrate to the active site is limited. In this situat ion, electrocatalytic half-wave potentials, E1/2, are similar for oxid ation of bulk succinate and reduction of bulk fumarate and coincide cl osely with the E-degrees' value assigned to the FAD. At 25-degrees-C a nd pH 7, the apparent K(M) for fumarate reduction is 0.16 mM, and k(ca t) is 840 s-1. Accordingly the second-order rate constant, k(cat)/K(M) , is 5.3 X 10(6) M-1 s-1. Under the same conditions, oxidation of succ inate is much slower. As the supply of fumarate to the enzyme is raise d to increase turnover, the electrochemical reaction eventually become s limited by the rate of electron transfer from the electrode. Under t hese conditions a second catalytic wave becomes evident, the E1/2 valu e of which corresponds to the reduction potential of the redox couple suggested to be center 2. This small boost to the catalytic current in dicates that the low-potential [4Fe-4S] cluster can function as a seco nd center for relaying electrons to the FAD.