PURIFICATION AND CHARACTERIZATION OF A NOVEL DIMERIC FERREDOXIN (FDIII) FROM RHODOBACTER-CAPSULATUS

A new ferredoxin, called FdIII, has been isolated and purified from th e photosynthetic bacterium Rhodobacter capsulatus. Its complete amino acid sequence has been determined. The FdIII polypeptide consists of 1 00 residues, including 9 cysteines and has a calculated molecular mass of 10,688 Da, which was confirmed by electrospray mass spectrometry. In its native form, FdIII is a homodimer as deduced from molecular sie ve chromatography and non-denaturing polyacrylamide gel electrophoresi s, as well as cross-linking experiments. The dimeric ferredoxin was fo und to contain 15.2 +/- 0.6 iron atoms and 13 +/- 1 inorganic sulfur a toms, consistent with the presence of four [4Fe-4S] clusters/molecule. The UV visible absorption spectrum of oxidized FdIII exhibited maxima at 282 and at 386 nm and a shoulder near 314 nm. FdIII was fully redu ced by excess dithionite at pH 8.0 or photochemically using 5-deazafla vin. Anaerobic oxidative titration of reduced FdIII with thionin indic ated that each FdIII monomer exchanges two electrons. Exposure of FdII I to air resulted in a rapid and irreversible oxidative denaturation o f the Fe-S clusters. The EPR spectrum of fully reduced FdIII showed a broad signal with an average g value of 1.94 that integrated to about two spins/monomer. EPR analysis of partially reduced FdIII (approximat ely 20% reduction) revealed a complex set of signals which was interpr eted as being the resulting sum of the contribution of two distinct pa ramagnetic centres. Based on its biochemical and spectroscopic propert ies, it is concluded that FdIII is a dimeric ferredoxin containing fou r [4Fe-4S] clusters. The synthesis of FdIII occurs only under growth c onditions allowing the derepression of nif genes. Results of in vitro electron transfer assays indicate that FdIII cannot serve as an electr on donor to nitrogenase.