Characterisation of oxidised 7Fe dicluster ferredoxins with NMR spectroscopy.
Dicluster ferredoxins (Fds) from Sulfolobus acidocaldarius and Desulfovibrio africanus (FdIII) have been studied using 1H NMR. Both wild-type proteins contain a [3Fe-4S]+/0 and a [4Fe-4S]2+/+ cluster as isolated. The [4Fe-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 transformation from the 7Fe form to the 8Fe form, but S. acidocaldarius Fd does not. Many factors determine the propensity of a cluster to undergo interconversion, including the presence, and correct orientation, of a suitable ligand. We have investigated this using 1H NMR by introducing a potential fourth ligand into the binding motif of cluster I of D. africanus FdIII. Asp14 has been mutated to cysteine (D14C), glutamic acid (D14E) and histidine (D14H). 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 1H NMR chemical shifts of mono- and di-cluster [3Fe-4S]+ cluster-containing ferredoxins is given.
Hannan
J P
JP
School of Chemical Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
Busch
J L
JL
Breton
J
J
James
R
R
Thomson
A J
AJ
Moore
G R
GR
Davy
S L
SL
D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000737:chemistry; D003901:Desulfovibrio / Q000737:chemistry; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D008667:Metalloproteins; D019159:Nonheme Iron Proteins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016945:Sulfolobus acidocaldarius / Q000737:chemistry
D019843:Archaeal Proteins; D005288:Ferredoxins; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins