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 extremely electroactive state at an electrode, with retention of native catalytic properties. The membrane-extrinsic FrdAB component adsorbs to monolayer coverage at edge-oriented pyrolytic graphite and catalyzes reduction of fumarate or oxidation of succinate, depending upon the electrode potential. In the absence of substrates, reversible redox transformations of centers in the enzyme are observed by cyclic voltammetry. The major component of the voltammograms is a pair of narrow reduction and oxidation signals corresponding to a pH-sensitive couple with formal reduction potential E degree' = -48 mV vs SHE at pH 7.0 (25 degrees C). This is assigned to two-electron reduction and oxidation of the active-site FAD. A redox couple with E degree' = -311 mV at pH 7 is assigned to center 2 ([4Fe-4S]2+/1+). Voltammograms for fumarate reduction at 25 degrees C, measured with a rotating-disk electrode, show high catalytic activity without the low-potential switch-off that is observed for the related enzyme succinate dehydrogenase. The catalytic electrochemistry is interpreted in terms of a basic model incorporating mass transport of substrate, interfacial electron transfer, and intrinsic kinetic properties of the enzyme, each of these becoming a rate-determining factor under certain conditions. Electrochemical reversibility is approached under conditions of low turnover rate, for example, as the supply of substrate to the active site is limited. In this situation, electrocatalytic half-wave potentials, E1/2, are similar for oxidation of bulk succinate and reduction of bulk fumarate and coincide closely with the E degree' value assigned to the FAD. At 25 degrees C and pH 7, the apparent KM for fumarate reduction is 0.16 mM, and kcat is 840 s-1. Accordingly the second-order rate constant, kcat/KM, is 5.3 x 10(6) M-1 s-1. Under the same conditions, oxidation of succinate is much slower. As the supply of fumarate to the enzyme is raised to increase turnover, the electrochemical reaction eventually becomes limited by the rate of electron transfer from the electrode. Under these conditions a second catalytic wave becomes evident, the E1/2 value of which corresponds to the reduction potential of the redox couple suggested to be center 2. This small boost to the catalytic current indicates that the low-potential [4Fe-4S] cluster can function as a second center for relaying electrons to the FAD.