Abstract
The gene encoding Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000), a dimeric octaheme cytochrome belonging to the polyheme cytochrome c3 superfamily, has been cloned and successfully expressed in another sulfate reducing bacteria, D. desulfuricans G201. The gene, named cycD, is monocistronic and encodes a cytochrome precursor of 135 amino acids with an extension at the NH2 terminus of 24 amino acids. This extension acts as a signal sequence which allows export across the cytoplasmic membrane into the periplasmic space. Tyrosine 73, which is in a close contact with the histidine sixth axial ligand to the heme 4 iron atom, has been replaced by a glutamate residue using site-directed mutagenesis. The cytochrome mutant when expressed in D. desulfuricans G201, is correctly folded and matured. A global increase of the oxidoreduction potentials of about 50 mV is measured for the Y73E cytochrome. The mutation also has a strong influence on the interaction of the cytochrome with its redox partner, the hydrogenase. This suggests, like the tetraheme cytochrome c3 (Mr 13, 000), heme 4 is the interactive heme in the cytochrome-hydrogenase complex and that alteration of the heme 4 environment can greatly affect the electron transfer reaction with its redox partner.
Highlights
Multi-redox center proteins are involved in several reactions of energy transduction phenomena
Northern blot analyses revealed only one hybridization band when an intragenic cycD fragment was used as probe against total RNA isolated either from D. desulfuricans Norway (Fig. 2, lane B) or from D. desulfuricans G201(pCACC3) (Fig. 2, lane A)
We have reported the influence of replacement of an invariant aromatic residue on the physicochemical properties and activity of cytochrome c3 (Mr 26,000) from D. desulfuricans Norway
Summary
A 1.5-kb fragment obtained from a double EcoRV-SacI digestion of the genomic DNA was selected and subsequently cloned into pUCBM21 (Boehringer Mannheim) cut with the same enzymes to obtain pUCC20 Both the 1.5-kb BamHI fragment from pUCC19 and the 1.5-kb EcoRI-SacI fragment from pUCC20 were subcloned into the replicative form of M13mp cut with the corresponding enzymes to give mp18C19 and mp18C20, respectively. Broad-host Range Plasmid Construction and Bacterial Conjugation—The replicative forms of mp18CACC3 and mp18CC3Y73E were digested with both EcoRI and HindIII and the resulting 895-bp fragments were ligated to the pJRD215 vector previously digested with the same enzymes to give pCACC3 and pCC3Y73E, respectively. Sulting plasmids were transformed into E. coli DH5␣ and subsequently transferred to D. desulfuricans G201 as described previously [21] Purification of Both Cytochrome c3 (Mr 26,000) and Y73E Cytochrome Mutant Expressed in D. desulfuricans G201—D. desulfuricans G201(pCACC3) and D. desulfuricans G201(pCC3Y73E) cells were obtained from 300-liter fermentations in Postgate C medium supplemented with 0.28 mM kanamycin. Both cytochromes were diluted in 50 mM Hepes buffer (pH 7.5) and injected using a flow rate of 30 l/min (150 l)
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