Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous oxide reductase from denitrifying Pseudomonas stutzeri

Abstract

The nos genes of Pseudomonas stutzeri are required for the anaerobic respiration of nitrous oxide, which is part of the overall denitrification process. A nos-coding region of ca. 8 kilobases was cloned by plasmid integration and excision. It comprised nosZ, the structural gene for the copper-containing enzyme nitrous oxide reductase, genes for copper chromophore biosynthesis, and a supposed regulatory region. The location of the nosZ gene and its transcriptional direction were identified by using a series of constructs to transform Escherichia coli and express nitrous oxide reductase in the heterologous background. Plasmid pAV5021 led to a nearly 12-fold overexpression of the NosZ protein compared with that in the P. stutzeri wild type. The complete sequence of the nosZ gene, comprising 1,914 nucleotides, together with 282 nucleotides of 5'-flanking sequences and 238 nucleotides of 3'-flanking sequences was determined. An open reading frame coded for a protein of 638 residues (Mrs 70,822) including a presumed signal sequence of 35 residues for protein export. The presequence is in conformity with the periplasmic location of the enzyme. Another open reading frame of 2,097 nucleotides, in the opposite transcriptional direction to that of nosZ, was excluded by several criteria from representing the coding region for nitrous oxide reductase. Codon usage for nosZ of P. stutzeri showed a high G+C content in the degenerate codon position (83.9% versus an average of 60.2%) and relaxed codon usage for the Glu codon, characteristic features of Pseudomonas genes from other species. E. coli nitrous oxide reductase was purified to homogeneity. It had the Mr of the P. stutzeri enzyme but lacked the copper chromophore.

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

“…This similarity was also shown by magnetic circular dichroism (MCD) [49,50] and extended X-ray absorption fine structure (EXAFS) studies, as the curve-fitted copper EXAFS results for N 2 OR are strikingly similar to those for COX [50][51][52]. Finally, the identification of a set of potential copper ligands in the C-terminal domain of N 2 OR, which matched the ones of the CuA center in COX, was crucial to establish that the CuA center, initially believed to be unique to COX, is also present in N 2 OR [53,54] (Fig. 1, Panel A).…”

Spectroscopic and Mutagenesis Studies on the CuA Centre from The Cytochrome‐c Oxidase Complex of Paracoccus Denitrificans

“…The spectrum of Cu, in bovine heart cytochrome-c oxidase has been obtained by two independent methods, namely by difference spectroscopy between different ligated forms using the magnetisation characteristics (field and temperature dependence) of each feature as a guide to its assignment (Greenwood et al, 1983), and from the MCD of an orientated sample (Greenwood et al, 1988). The Tn5 mutant form of nitrous-oxide reductase is deficient in copper, containing up to two copper atomddimer instead of the eight found in the wild-type enzyme and has been shown to contain only the Cu, centre (Viebrock and Zumft, 1987;Dooley et al, 1991 ;Antholine et al, 1992). The spectra of the three species are closely similar in the region 400-900 nm.…”

“…Nitrous-oxide reductase was purified from strain MK402 of Pseudomonas stutzeri, which is defective in copper chromophore biosynthesis (Viebrock and Zumft, 1987;Zumft et al, 1990). The bacteria were grown in wild-type medium (Coyle et al, 1985) supplemented with kanamycin and streptomycin.…”

A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri)

“…The enzyme responsible for catalysing this 2-electron reduction is nitrous oxide reductase (N,OR), which is composed of 2 identical subunits (M, -70 000) of known amino acid sequence [2] and has a stoichiometry of about 4 copper ions per monomer [3]. A variety of spectroscopic methods have been applied to the study of the copper ccntres but no clear structural model has yet been proposed for them.…”