Pauleta, Sr., F. Guerlesquin, C. F. Goodhew, B. Devreese, J. VanBeeumen, AS Pereira, I. Moura, and G. W. Pettigrew. "
Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase."
Biochemistry. 43 (2004): 11214-11225.
AbstractThe gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.
Almendra, MJ, CD Brondino, O. Gavel, AS Pereira, P. Tavares, S. Bursakov, R. Duarte, J. CALDEIRA, JJG Moura, and I. Moura. "
Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas."
Biochemistry. 38 (1999): 16366-16372.
AbstractAn air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein, Selenium was not detected. The UV/visible absorption spectrum of D, gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with Fe-57 and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.
Pereira, AS, C. G. Timoteo, M. Guilherme, F. Folgosa, S. G. Naik, A. G. Duarte, BH HUYNH, and P. Tavares. "
Spectroscopic Evidence for and Characterization of a Trinuclear Ferroxidase Center in Bacterial Ferritin from Desulfovibrio vulgaris Hildenborough."
Journal of the American Chemical Society. 134 (2012): 10822-10832.
AbstractFerritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of similar to 80 angstrom in diameter. The main function of ferritin is to oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe2+ (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe2+, and the products were characterized by Mossbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe2+Fe3+ species, and a mononuclear Fe2+ species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a mu-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe2+Fe3+ state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.