WUNSCH P, HERB M, WIELAND H, SCHIEK UM, ZUMFT WG
Requirements for CuA and
Cu-S center assembly of nitrous oxide reductase deduced from complete
periplasmic enzyme maturation in the nondenitrifier Pseudomonas putida.
J Bacteriol 185: 887-896 (2003)
Bacterial nitrous oxide (N2O) reductase is the terminal
oxidoreductase of a respiratory process that generates dinitrogen from N2O.
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 CuA
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 N2O 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-N2O
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 CuA metallation. Both of these were found to be
dispensable elements for N2O reductase biosynthesis. Our
experimental and in silico data were integrated in a model of N2O
reductase maturation.