DISCOVERY OF A NOVEL FERREDOXIN FROM AZOTOBACTER-VINELANDII CONTAINING 2 [4FE-4S] CLUSTERS WITH WIDELY DIFFERING AND VERY NEGATIVE REDUCTION POTENTIALS

Ferredoxins that contain 2[4Fe-4S](2+/+) clusters can be divided into two classes. The ''clostridial-type'' ferredoxins have two Cys-Xaa-Xaa -Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro motifs. The ''chromatium-type'' f erredoxins have one motif of that type and one more unusual Cys-Xaa-Xa a-Cys-Xaa(7-9)-Cys-Xaa-Xaa-Xaa-Cys-Pro motif. Here we report the purif ication of a novel ferredoxin (FdIII) from Azotobacter vinelandii whic h brings to 12 the number of small [Fe-S] proteins that have now been reported from this organism. NH2-terminal sequencing of the first 56 a mino acid residues shows that FdIII is a chromatium-type ferredoxin wi th 77% identity and 88% similarity to Chromatium vinosum ferredoxin. S tudies of the purified protein by matrix-assisted laser desorption ion ization-time of flight mass spectroscopy, iron analysis, absorption, c ircular dichroism, and electron paramagnetic resonance spectroscopies show that FdIII contains 2[4Fe-4S](2+/+) clusters in a 9,220-Da polype ptide. All 2[4Fe-4S](2+/+) ferredoxins that have been studied to date, including C. vinosum ferredoxin, are reported to have extremely simil ar or identical reduction potentials for the two clusters. In contrast , electrochemical characterization of FdIII clearly establishes that t he two [4Fe-4S](2+/+) clusters have very different and highly negative reduction potentials of -486 mV and -644 mV versus the standard hydro gen electrode.