Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus

A ferredoxin was purified anaerobically from Rhodobacter capsulatus grown photoheterotrophically with excess ammonia. This ferredoxin, called ferredoxin II (FdII), had a molecular weight of approximatively 15,000 by gel filtration and 14,000 by SDS polyacrylamide gel electrophoresis indicating that it is monomeric. Its absorption spectrum (oxidized form) exhibited maxima at 280 nm and 400 nm; the A400/A280 ratio had a calculated value of 0.55. Chemical determination of its iron and sulfur atom content, the value of the extinction coefficient at 400 nm (epsilon 400 = 26.8 mM-1 cm-1) and EPR spectra indicated that ferredoxin II contained one 3Fe-4S] and one 4Fe-4S] cluster. Upon reduction with excess dithionite only the 3Fe-4S] cluster became reduced. The reduction of both clusters was achieved by using 5-deazaflavin as photocatalyst. Ferredoxin II was also purified from bacteria grown under nitrogen limiting (nif derepressing) conditions. In in vitro assays, ferredoxin II catalyzed electron transport between illuminated chloroplasts and nitrogenase.