Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum.
The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.
D003013:Clostridium / Q000378:metabolism*; D004396:Coloring Agents / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D005288:Ferredoxins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism; D010640:Phenothiazines / Q000494:pharmacology; D012643:Selenium / Q000494:pharmacology*
D004396:Coloring Agents; D005288:Ferredoxins; D010640:Phenothiazines; C026518:apoferredoxin; D004227:Dithionite; D010088:Oxidoreductases; D006864:Hydrogenase; D012643:Selenium; C009469:thionine