Chromate reductase activity of the Paracoccus denitrificans ferric reductase B (FerB) protein and its physiological relevance.
The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53 ± 0.09 (O(2) absent) or > 2 (O(2) present), the apparent K (M) value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s(-1) (mg protein)(-1). Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.
Sedláček
Vojtěch
V
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 62500, Brno, Czech Republic.
Kučera
Igor
I
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002840:Chromates / Q000378:metabolism*; D038181:FMN Reductase / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010101:Oxygen Consumption; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D002840:Chromates; D005420:Flavoproteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D010088:Oxidoreductases; D038181:FMN Reductase; C023903:ferric citrate iron reductase; C063243:chromate reductase