A RHIZOBIUM-MELILOTI FERREDOXIN (FDXN) PURIFIED FROM ESCHERICHIA-COLIDONATES ELECTRONS TO RHODOBACTER-CAPSULATUS NITROGENASE

The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferred oxin (FdxN) was expressed in Escherichia coil under the control of the lac promoter. The fdxN gene product was purified under anaerobic cond itions by ion-exchange chromatography and gel-filtration steps using a n antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predi cted R. meliloti FdxN protein in its amino acid composition and N-term inal amino acid sequence. Chemical determination of the iron content r evealed 8.6+/-0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A(378)/A(284) ratio of 0.7. EPR spectroscop y revealed a rhombic signal when FdxN was partially reduced, and a bro ad signal indicative of spin-spin interaction when fully reduced, sugg esting the presence of two Fe-S clusters/ferredoxin polypeptide. Our d ata suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN wa s able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.