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-ISOMERASEGENE

The single conserved Cys165 outside of the Cu-A domain of nitrous oxid e reductase (N(2)OR) from Pseudomonas stutzeri was mutated to glycine to test its presumed function in metal coordination of the catalytic s ite, Cu-z. The point mutation reduced the cellular level of N(2)OR 5-1 0-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 m utant enzyme was processed and exported into the periplasm like the wi ld-type enzyme. Chemical analysis for sulfhydryl groups gave about nin e -SH groups/monomer of the apoenzyme prepared from the wild-type enzy me, in accordance with the nine cysteine residues of the derived a!nin o acid sequence. Eight -SH groups were found to form disulfide bridges in the holoenzyme dimer. We propose that in the native state of the e nzyme Cys165 does not bind to Cu-z, but may be part of a disulfide bri dge essential fur the stability of N(2)OR. Immediately downstream of t he genes nosDFY, encoding the components for Cu incorporation into the reductase, we have identified the open reading frame, ORFL, whose der ived product has the signature of a protein disulfide isomerase.