Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome cd 1 Biosynthesis in Pseudomonas stutzeri

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Abstract
By transforming N 2 O to N 2 , 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 N 2 O 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 Cu A 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 N 2 O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome cd 1 nitrite reductase was found in the periplasm of the tatC mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D 1 macrocycle. The NirD protein as part of a complex required for heme D 1 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.

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