Carla Ascenso

Desulfoferrodoxin: a modular protein.

Abstract. The gene encoding the non-heme iron-containing desulfoferrodoxin from Desulfovibrio vulgaris was cloned in two fragments in order to obtain polypeptides corresponding to the N- and C-terminal domains observed in the tertiary structure. These fragments were expressed in Escherichia coli, purified to homogeneity and biochemically and spectroscopically characterized. Both recombinant fragments behaved as independent metal-binding domains. The N-terminal fragment exhibited properties similar to desulforedoxin, as expected by the presence of a Fe(S-Cys)4 metal binding motif. The C-terminal fragment, which accommodates a Fe(Nε-His)3(Nδ-His)(S-Cys) center, was shown to have properties similar to neelaredoxin, except for the reaction with superoxide. The activities of desulfoferrodoxin and of the expressed C-terminal fragment were tested with superoxide in the presence and absence of cytochrome c. The results are consistent with superoxide reductase activity and a possible explanation for the low superoxide consumption in the superoxide dismutase activity assays is proposed.

The use of 113Cd NMR chemical shifts as a structural probe in tetrathiolate metalloproteins

Abstract A wide range of 113Cd chemical shifts has been observed for 113Cd-substituted metalloproteins ranging from −100 ppm, for Cd with octahedral oxygen ligands, to +760 ppm for tetrahedral sulfur ligands. In particular the 113Cd chemical shifts of tetrahedral sulfur bound sites, for proteins such as rubredoxin and desulforedoxin, appear around 720–745 ppm. New 113Cd chemical shift data for 113Cd-substituted, overexpressed and mutated homologous desulforedoxin-like Fe(S-Cys)4 proteins, have been obtained and a correlation between the 113Cd chemical shift and structure at the metal site has been observed. This subtle effect of geometry at the metal centre on 113Cd chemical shifts can be explained in terms of an increase in the paramagnetic term for the chemical shift of the 113Cd nucleus as distortion of the tetrathiolate centre is increased.

Superoxide reductase activities of neelaredoxin and desulfoferrodoxin metalloproteins

Superoxide reductases have now been well characterized from several organisms. Unique biochemical features include the ability of the reduced enzyme to react with O2- but not dioxygen (reduced SORs are stable in an aerobic atmosphere for hours). Future biochemical assays that measure the reaction of SOR with O2- should take into account the difficulties of assaying O2- directly and the myriad of redox reactions that can take place between components in the assay, for example, direct electron transfer between cytochrome c and Dfx. Future prospects include further delineation of the reaction mechanisms, characterization of the putative (hydro)peroxo intermediate, and studies that uncover the components between reduced pyridine nucleotides and SOR in the metabolic pathway responsible for O2- detoxification.

Zinc-substituted Desulfovibrio gigas desulforedoxins: Resolving subunit degeneracy with nonsymmetric pseudocontact shifts

Desulfovibrio gigas desulforedoxin (Dx) consists of two identical peptides, each containing one [Fe‐4S] center per monomer. Variants with different iron and zinc metal compositions arise when desulforedoxin is produced recombinantly from Escherichia coli. The three forms of the protein, the two homodimers [Fe(III)/Fe(III)]Dx and [Zn(II)/Zn(II)]Dx, and the heterodimer [Fe(III)/Zn(II)]Dx, can be separated by ion exchange chromatography on the basis of their charge differences. Once separated, the desulforedoxins containing iron can be reduced with added dithionite. For NMR studies, different protein samples were prepared labeled with 15N or 15N + 13C. Spectral assignments were determined for [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx from 3D 15N TOCSY‐HSQC and NOESY‐HSQC data, and compared with those reported previously for [Zn(II)/Zn(II)]Dx. Assignments for the 13Cα shifts were obtained from an HNCA experiment. Comparison of 1H–15N HSQC spectra of [Zn(II)/Zn(II)]Dx, [Fe(II)/Fe(II)]Dx and [Fe(II)/Zn(II)]Dx revealed that the pseudocontact shifts in [Fe(II)/Zn(II)]Dx can be decomposed into inter‐ and intramonomer components, which, when summed, accurately predict the observed pseudocontact shifts observed for [Fe(II)/Fe(II)]Dx. The degree of linearity observed in the pseudocontact shifts for residues ≥8.5 Å from the metal center indicates that the replacement of Fe(II) by Zn(II) produces little or no change in the structure of Dx. The results suggest a general strategy for the analysis of NMR spectra of homo‐oligomeric proteins in which a paramagnetic center introduced into a single subunit is used to break the magnetic symmetry and make it possible to obtain distance constraints (both pseudocontact and NOE) between subunits.

Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides

Abstract Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)4 metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe3+, Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellmans reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.

NMR solution structures of two mutants of desulforedoxin.

The differences in geometry at the metal centres in the two known [Fe-4S] proteins rubredoxin (Rd) and desulforedoxin (Dx) are postulated to be a result of the different spacing of the C-terminal cysteine pair in the two proteins. In order to address this question, two mutants of Desulfovibrio gigas Dx with modified cysteinyl spacing were prepared and their solution structures have been determined by NMR. Mutant 1 of Dx (DxM1) has a single glycine inserted between the adjacent cysteines (C28 and C29) found in the wild type Dx sequence. Mutant 3 (DxM3) has two amino acid residues, -P-V-, inserted between C28 and C29 in order to mimic the primary sequence found in Rd from Desulfovibrio gigas. The solution structure of DxM1 exists, like wild type Dx, as a dimer in solution although the single glycine inserted between the adjacent cysteines disrupts the stability of the dimer resulting in exchange between a dimer state and a small population of another, probably monomeric, state. For DxM3 the two amino acid residues inserted between the adjacent cysteines results in a monomeric protein that has a global fold near the metal centre very similar to that found in Rd.