Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S](2+/ 1+) cluster and one [3Fe-4S](1+/0) cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys(11 )-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)- Cys(51)-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S] tt[4Fe-4S] cluster interconversion with a wide range of metal ions. In orde r to obtain protein for mutagenesis studies Da FdIII has been cloned, seque nced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escheric hia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether transla ted from a synthetic gene (pJB10) or from the native nucleotide sequence (p JB11), occurred at similar levels (approx. 6 mg . l(-1)), but without incor poration of metal clusters. The nucleotide sequence confirms the protein se quence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikia n (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achi eved using FeCl3 together with cysteine sulphur transferase, NifS, plus cys teine to generate low levels of sulphide ions. Absorption and EPR spectrosc opy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. T hin-film electrochemistry provides evidence that the [3Fe-4S] cluster under goes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the pro tein has lower stability than native Da FdIII during chromatography. The on e-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructu red protein with random coil chemical shifts whereas spectra of the reconst ituted ht protein show secondary structural elements and 18 peaks shifted d ownfield of 9.6 p.p.m. The spectra are unique but have similarities with th e shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does no t affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of th e ht compared with the native protein.