Cristina G. Timóteo

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

Ferritins 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 ~80 A in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (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 Fe(2+), 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 Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) 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 Fe(2+)Fe(3+) 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.