Requirements for Cu A and Cu-S Center Assembly of Nitrous Oxide Reductase Deduced from Complete Periplasmic Enzyme Maturation in the Nondenitrifier Pseudomonas putida

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Abstract
Bacterial nitrous oxide (N 2 O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N 2 O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear Cu A site in the periplasm. There are seven putative maturation factors, encoded by nosA , nosD , nosF , nosY , nosL , nosX , and sco . We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida . An in silico study of denitrifying bacteria revealed that nosL , nosX (or a homologous gene, apbE ), and sco , but not nosA , coexist consistently with the N 2 O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N 2 O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri , we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for Cu A metallation. Both of these were found to be dispensable elements for N 2 O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N 2 O reductase maturation.