PURIFICATION AND CHARACTERIZATION OF A FIXABCX-LINKED 2[4FE-4S] FERREDOXIN FROM AZOTOBACTER-VINELANDII

Ferredoxins that contain 2[4Fe-4S](2+/+) clusters can be divided into two classes. The ''clostridial-type'' ferredoxins have two CysXXCysXXC ysXXXCysPro motifs. The ''photosynthetic bacterial and nif-related'' f erredoxins have one motif of that type and one more unusual CysXXCysX( 7-9)CysXXXCysPro motif. In Azolobacter vinelandii three gene sequences have been reported that contain the latter motif, but until now none of the gene products has been purified. Here we report the purificatio n of a small anionic [Fe-S] pro rein with yields of similar to 3 mg pe r 500 g cell paste. NH2-terminal sequence analysis shows that this pro tein is the product of a previously sequenced A. vinelandii gene that is found upstream of fixA and is cotranscribed with fixABCX. That gene was originally named fixP, but since that gene designation is now com monly used for a very different cb-type cytochrome oxidase we have ren amed the gene fixFd and its product Fix Fd. Its sequence places Fix Fd in the class of ''photosynthetic bacterial and nif-related'' 2[4Fe-4S ](2+/1+) ferredoxins that includes Chromatium vinosum ferredoxin. Stud ies of the purified protein by Fe analysis, absorption, CD and EPR spe ctroscopies and electrochemistry confirm this characterization; the re duction potentials of the two clusters are -440 mV vs SHE. The fact th at A. vinelandii synthesizes three different proteins with the same se quence motif, each of which is likely to have a different function, sh ows that although sequence motifs may be used reliably to classify fer redoxins by cluster type they cannot yet be used reliably for classify ing ferredoxins by function.