Mutation of the conserved Cys165 outside of the Cu-A domain destabilizes nitrous oxide reductase but maintains its catalytic activity - Evidence for disulfide bridges and a putative protein disulfide isomerase gene

The single conserved Cys165 outside of the Cu-A domain of nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic site, Cu-z. The point mutation reduced the cellular level of N(2)OR 5-10-fold compared to the level of the control strain. In the mutant, the activity and the Cu content of the enzyme, as well as the transcript level of the N(2)OR structural gene, nosZ, remained unaffected.. The mutant enzyme was processed and exported into the periplasm like the wild-type enzyme. Chemical analysis for sulfhydryl groups gave about nine -SH groups/monomer of the apoenzyme prepared from the wild-type enzyme, in accordance with the nine cysteine residues of the derived a!nino acid sequence. Eight -SH groups were found to form disulfide bridges in the holoenzyme dimer. We propose that in the native state of the enzyme Cys165 does not bind to Cu-z, but may be part of a disulfide bridge essential fur the stability of N(2)OR. Immediately downstream of the genes nosDFY, encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose derived product has the signature of a protein disulfide isomerase.