Dicluster ferredoxins (Fds) from Sulfolobus acidocaldarius and Desulfovibri
o africanus (FdIII) have been studied using H-1 NMR. Both wild-type protein
s contain a [3Fe-4S](+/0) and a [4Fe-4S](2+/+) cluster as isolated. The [4F
e-4S](2+/+) cluster (cluster II) is bound by cysteine residues arranged in
a classic ferredoxin motif: CysI-(Xaa)(2)-CysII- (Xaa)(2)-CysIII- (Xaa)(n)-
CysIV-Pro, whilst the binding motif of the [3Fe-4S](+/0) cluster (cluster I
) has a non-ligating aspartic acid (Asp14) at position II, i.e. CysI-(Xaa)(
2)-Asp- (Xaa)(2)-CysIII. D. africanus FdIII undergoes facile cluster transf
ormation from the 7Fe form to the 8Fe form, but S. acidocaldarius Fd does n
ot. Many factors determine the propensity of a cluster to undergo interconv
ersion, including the presence, and correct orientation, of a suitable liga
nd. We have investigated this using 1H NMR by introducing a potential fourt
h ligand into the binding motif of cluster I of D. africanus FdIII. Asp14 h
as been mutated to cysteine (D14C), glutamic acid (D14E) and histidine (D14
H). Cluster incorporation was performed in vitro. The cluster types present
were identified from the chemical shift patterns and temperature-dependent
behaviour of the hyperfine-shifted resonances. Factors influencing cluster
ligation and cluster interconversion, in vitro, are discussed. Furthermore
, the data have established that the residue at position II in the cluster
binding motif of cluster I is influential in determining the chemical shift
pattern observed for a [3Fe-4S](+) cluster when a short/symmetric binding
motif is present. Based on this, a series of rules for characterising the H
-1 NMR chemical shifts of mono- and di-cluster [3Fe-4S](+) cluster-containi
ng ferredoxins is given.