Sharon L. Davy

PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity

PrrC from Rhodobacter sphaeroides provides the signal input to a two‐component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052‐2062; Oh, J.‐I. and Kaplan, S. (2000) EMBO J. 19, 4237‐4247). It is also a homologue of eukaryotic Sco proteins and each has a C‐x‐x‐x‐C‐P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase. Overexpression and purification of a water‐soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal‐free rPrrC (apo‐PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV‐visible spectrum, having absorbance with a λ max of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal‐free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper‐loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol‐disulfide oxidoreductase function and a copper‐binding role.

Determination of the [Fe4S4]Cys4 cluster geometry of Desulfovibrio africanus ferredoxin I by 1H NMR spectroscopy

1D and 2D 1H NMR studies of the Fe4S4, cluster containing ferredoxin I from Desulfovibrio africanus have been carried out with the aim of determining the geometry of the cluster linkages with the 4 Cys side chains that bind the cluster. This required the Cys βCH resonances of the oxidised protein to be sequence‐specifically and stereo‐specifically assigned, and this was accomplished by a combination of TOCSY and NOE measurements, allied to model building based on X‐ray structures of related ferredoxins. An analysis of the estimated hyperfine shifts of the Cys βCH resonances with a Karplus‐type equation relating the shifts to iron‐sulfur‐β carbon‐/3 proton dihedral angles, taken together with the relative relaxation rates of the two βCH2 resonances, estimated from their linewidths, then allowed the ironsulfur‐/3‐carbon‐α‐carbon dihedral angles to be determined. A novel representation of the NMR data is presented which shows that the cluster dihedral angles are uniquely determined by the NMR data. The analysis reveals that the dihedral angles for D. africanus ferredoxin I are similar to the corresponding angles of other ferrredoxins even though there are differences in their 1H NMR spectra. The sequence‐specific and stereospecific assignments have been extended by analogy to the related Fe4S4‐containing D. gigas ferredoxin I, and the stereospecific assignments to the Fe4S4‐containing Thermococcus litoralis ferredoxin.

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus

The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene (bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18,174 Da. The molecular mass of the purified protein was estimated to be 18,176. +/ 0.80 Da by electrospray mass spectrometry. The bfr was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conversed in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.

Effect of nickel(II) substitution on the resonance Raman and NMR spectra of Alcaligenes xylosoxidans azurin II: implications for axial-ligand bonding interactions in cupredoxin active sites

Abstract Assignment of the resonance Raman (RR) spectrum of Ni(II)-substituted azurin II from Alcaligenes xylosoxidans (NCIMB 11015) using Ni isotope substitution reveals an anomalously low Ni-S(Cys) stretching frequency of 349 cm–1, suggesting the presence of significant axial-ligand bonding interactions. The X-ray crystal structure of Ni(II)-substituted azurin from Pseudomonas aeruginosa shows that there are two potential axial ligands to the Ni ion: a peptide carbonyl O at a distance of 2.46 Å, together with a long-range interaction from a methionine sulfur (S′) at a distance of 3.30 Å. Comparison of the RR properties of Ni(II)-substituted azurin II with stellacyanin (which contains an axial carbonyl ligand, but no methionine) suggests that the interaction from the carbonyl oxygen ligand alone is not sufficient to account for the weak Ni azurin metal-thiolate bond. Instead, it appears that a Ni-methionine bonding interaction is also required to explain the low Ni-S(Cys) stretching frequency in Ni(II)-substituted azurin II. This hypothesis is supported by NMR studies which show a large paramagnetic shift for the protons of the methionine side-chain. Thus, it appears that Ni-substituted azurin II is best described as five-coordinate, and that significant Ni(II)-methionine bonding interactions can occur at a distance of 3.3 Å.

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1H-NMR experiments and both alpha-helix and beta-sheet structure detected. The influence of the paramagnetism of the Fe4 S4 cluster on the NMR properties of the ferredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

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]<-->[4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg.l(-1)), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.

Slow formation of [3Fe–4S]1+ clusters in mutant forms of Desulfovibrio africanus ferredoxin III

Desulfovibrio africanus ferredoxin III (Da FdIII) readily interconverts between a 7Fe and an 8Fe form with Asp‐14 believed to provide a cluster ligand in the latter form. To investigate the factors important for cluster interconversion in Fe/S cluster‐containing proteins we have studied two variants of Da FdIII produced by site‐directed mutagenesis, Asp14Glu and Asp14His, with cluster incorporation performed in vitro. Characterisation of these proteins by UV/visible, EPR and 1H NMR spectroscopies revealed that the formation of the stable 7Fe form of these proteins takes some time to occur. Evidence is presented which indicates the [4Fe–4S]2+ cluster is incorporated prior to the [3Fe–4S]1+ cluster.

Characterisation of oxidised 7Fe dicluster ferredoxins with NMR spectroscopy.

Abstract 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.