Purification and characterization of ferric reductases from Paracoccus denitrificans
| Autoři | |
|---|---|
| Rok publikování | 2002 |
| Druh | Článek ve sborníku |
| Konference | XVIII. BIOCHEMICKÝ ZJAZD |
| Fakulta / Pracoviště MU | |
| Citace | |
| Obor | Biochemie |
| Klíčová slova | ferric reductase; Paracoccus denitrificans; purification |
| Popis | Iron is a component of a number of biological systems, e.g. electron transport chains, cofactors of enzymes, regulatory proteins etc. It occurs in ferric form in environment, while organisms require ferrous iron. Enzymes catalysing the reduction of ferric complexes are widely spread within bacterial kingdom. Some of these enzymes are associated with cytoplasmic membrane, some are present in soluble form in cytoplasm or periplasm, some are excreted into extracellular medium. In our work, cellular fractions of Paracoccus denitrificans were tested for their activity towards reduction of various ferric complexes with NADH as an electron donor. We found the most of the ferric reductase activity in cytosolic fraction. Fe(III)-nitrilotriacetate was chosen as the best artificial substrate for activity measurements. Two proteins responsible for the activity was identified. Both enzymes were purified to homogeneity by combination of FPLC ion-exchange chromatography and chromatofocusing, and characterised with respect to kinetics of enzyme reaction. One of these enzymes (denoted FerB) does not need FMN or other flavins for its catalytic function. It differs in this aspect from other known ferric reductases which are considered to be in fact flavin reductases (Fontecave et al., 1994). Both NADH and NADPH serve as electron donors for FerB whereas FerA uses NADH exclusively. Mr of native enzymes were estimated (GPC) to be about 25 kDa and 55 kDa for FerA (pI 6.9) and FerB (pI 5.5), respectively. N-terminal sequences of FerA, FerB were determined and database searches revealed that the sequence of FerB is homologous to chromate reductase of Pseudomonas putida. Indeed, FerB has chromate reductase activity. |
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