NITRIC-OXIDE REDUCTASE FROM PSEUDOMONAS-STUTZERI - PRIMARY STRUCTURE AND GENE ORGANIZATION OF A NOVEL BACTERIAL CYTOCHROME BC COMPLEX

Nitric oxide (NO) reductase is an integral membrane component of the a naerobic respiratory chain of Pseudomonas sturzeri that transforms nit rate to dinitrogen (denitrification). The enzyme catalyzes the reducti on of NO to nitrous oxide. The structural genes for the NO reductase c omplex, norC and norB, were sequenced and their organization establish ed by primer extension and Northern blot analysis. The norCB genes enc oding the cytochrome c and cytochrome b subunits of the enzyme are con tiguous and transcribed as a single 2.0-kb transcript. The promoter re gion has a canonical recognition motif for the transcriptional activat or protein Fnr centered at -40.5 nucleotides from the initiation site of transcription. No similarity of the derived gene products to known cytochromes of b- or c-type was found in a data bank search. Post-tran slational processing of the two subunits was limited to the removal of the terminal methionine to leave an N-terminal serine in either subun it. The mature cytochrome c subunit (16508Da, 145 residues) is predict ed to be a bitopic protein with a single membrane anchor. The mature c ytochrome b subunit (53006Da, 473 residues) is a putatively polytopic, strongly hydrophobic membrane-bound protein with 12 potential transme mbrane segments. Several histidine and proline residues were identifie d with potentially structural and/or functional importance. Mutational inactivation of NO reductase by deletion of norB or the norCB genes a ffected strongly the in vivo activity of respiratory nitrite reductase (cytochrome cd(1)) to a much lesser extent the expression level of th is enzyme. In turn, mutational inactivation of the structural gene for cytochrome cd(1), nirS, or loss of in vivo nitrite reduction by mutat ion of the nirT gene, encoding a presumed tetraheme cytochrome, lowere d the expression level of NO reductase to 5-20%, but hardly its cataly tic activity. The cellular concentration of NO reductase increased aga in on restoration of nitrite reduction in the nirS=TnS mutant MK202 by complementation with nirS or with the heterologous nirK gene, encodin g the Cu-containing nitrite reductase from Pseudomonas aureofaciens. T hus, NO may be required as an inducer for its own reductase. Our resul ts show that the nitrite-reducing system and the NO-reducing system ar e not operating independently from each other but are interlaced by ac tivity modulation and regulation of enzyme synthesis.