B. Reyntjens et al., PURIFICATION AND CHARACTERIZATION OF A FIXABCX-LINKED 2[4FE-4S] FERREDOXIN FROM AZOTOBACTER-VINELANDII, JBIC. Journal of biological inorganic chemistry, 2(5), 1997, pp. 595-602
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.