Role of the Tat ransport system in nitrous oxide reductase translocation and cytochrome cd1 biosynthesis in Pseudomonas stutzeri.
By transforming N2O to N2, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified tat genes of Pseudomonas stutzeri and functionally analyzed the unlinked tatC and tatE loci. A tatC mutant retained N2O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center CuA rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N(2)O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a tatE mutant and a reductase-overproducing strain, we show a function for TatE in N2O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D1 macrocycle. The NirD protein as part of a complex required for heme D1 synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the tatC mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.
Heikkilä
M P
MP
Lehrstuhl für Mikrobiologie, Universität Karlsruhe, D-76128 Karlsruhe, Germany.
Honisch
U
U
Wunsch
P
P
Zumft
W G
WG
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D003470:Culture Media; D003574:Cytochrome c Group; D003580:Cytochromes / Q000096:biosynthesis*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009566:Nitrates / Q000378:metabolism; D009572:Nitrite Reductases / Q000096:biosynthesis*; D009573:Nitrites / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D010101:Oxygen Consumption; D011549:Pseudomonas / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D002352:Carrier Proteins; D003470:Culture Media; D003574:Cytochrome c Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D009566:Nitrates; D009573:Nitrites; C408585:twin-arginine translocase complex, E coli; D003300:Copper; C019315:cytochrome cd1; D010088:Oxidoreductases; D009572:Nitrite Reductases; C022763:nitrous oxide reductase