VOLTAMMETRIC CHARACTERIZATION OF RAPID AND REVERSIBLE BINDING OF AN EXOGENOUS THIOLATE LIGAND AT A [4FE-4S] CLUSTER IN FERREDOXIN-III FROM DESULFOVIBRIO-AFRICANUS

Rapid and reversible, redox-coupled binding of an exogenous ligand to an Fe-S cluster in a protein has been studied by voltammetry. Ferredoxin III from Desulfovibrio africanus contains a [3Fe-4S] cluster whose transformation into a [4Fe-4S] center generates a new site for coordination. By coadsorbing with neomycin or polymyxin, an electroactive film of the protein is obtained at an edge-oriented pyrolytic graphite electrode which is then introduced to solutions containing the ligand. The equilibria and kinetics of ligation are determined by inspection and digital simulation of voltammograms measured over a range of scan rates. For solutions of 2-mercaptoethanol, results are consistent with coordination of the thiolate anion at both oxidized (dissociation constant = 28 muM) and reduced (dissociation constant = 97 mM) forms of the transformed [4Fe-4S] cluster. Direct detection of the thiolate-ligated reduced cluster by EPR is not feasible because the high concentration of mercaptoethanol that is required in solution results in cluster degradation. No interaction with thiolate is detected for the indigenous, stable [4Fe-4S] cluster or for the untransformed [3Fe-4S] precursor cluster. Under fast-scan conditions, the thiolate-ligated species appears as a trapped redox couple with E-degrees' = -585 mV (cf. -396 mV for the native species) whereas, at a slow scan rate, equilibrium is established at all times and the observed reduction potential of the new couple depends upon thiolate concentration. At intermediate scan rates, a combination of trapped and dynamic, equilibrating species is observed. The great disparity in the equilibrium constants, reflected in the marked change in reduction potential, is attributed to a much faster rate of thiolate coordination to the oxidized cluster. An associative mechanism is proposed, involving nucleophilic attack by thiolate at the labile Fe subsite. The experiment provides a simple yet profound demonstration of time-separated coupling between electron transfer and ligand exchange (more generally extendable to conformational change) in a metalloprotein.