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

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.

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

“…(46). Since NosZ biosynthesis depends on auxiliary gene products that affect its expression level and protein translocation efficiency (8,13,18,47), we cloned the entire nos cluster, including the tatE gene for protein transport, into pUCP22, which resulted in the nosR expression vector pPRE-3 (Fig. 1A).…”

“…The plasmids pUC18 (Ap r ) (48) and pUCP22 (Ap r Gm r ) (46) were used as general cloning vectors. The plasmid pUCP22RZ was derived from pUCP22 and carries nosRZ within a 5.3-kb Eco47III-SmaI fragment (47); the plasmid pUCP22RE is another pUCP22 derivative which carries the entire nosRZDFYLtatE sequence within an 8.8-kb Eco47III-XbaI fragment (18). E. coli was grown in Luria-Bertani medium at 37°C.…”

“…Preparations of cell extract, the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the immunochemical detection of NosZ were described previously (47). His-tagged NosR proteins were detected immunochemically by use of an anti-His 5 antibody according to the procedures of the supplier (QIAGEN).…”

“…Plasmid pPR6hE was constructed from plasmid pUCP22RE (47) by the insertion of a 189-bp Kpn2I-Kpn2I PCR fragment into the Kpn2I site of pUCP22RE. The fragment was designed to add the codons for a His 6 tag, a stop codon, and a Kpn2I site at the 3Ј end of nosR.…”

“…Bacteria that use nitrous oxide as an electron sink for respiration synthesize a copper enzyme nitrous oxide (N 2 O) reductase (EC 1.7.99.6, encoded by the nosZ gene) and several ancillary proteins that participate in the biosynthesis of the copper centers Cu A and Cu Z (47,53). N 2 O utilization usually plays a part in nitrate or nitrite denitrification, but it also exists in nature as an autonomous respiratory mode, as exemplified in the genus Wolinella (50).…”