2 ISOFUNCTIONAL NITRIC-OXIDE REDUCTASES IN ALCALIGENES-EUTROPHUS H16

Two genes, norB and norZ, encoding two independent nitric oxide reduct ases have been identified in Alcaligenes eutrophas H16, norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%, While norB resides on the megaplasmid pHG1, the norZ gene is loca ted on a chromosomal DNA fragment, Amino acid sequence analysis sugges ts that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices, The region encompassing helices 3 to 14 sh ows similarity to the NorB subunit of common bacterial nitric oxide re ductases, including the positions of six strictly conserved histidine residues, Unlike the Nor enzymes characterized so far from denitrifyin g bacteria, NorB and NorZ of A, eutrophus contain an amino-terminal ex tension which may form two additional helices connected by a hydrophil ic loop of 203 amino acids, The presence of a NorB/NorZ-like protein w as predicted from the genome sequence of the cyanobacterium Synechocys tis sp, strain PCC6803, While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neig hboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs, The physiological roles of norB and norZ in A , eutrophus were investigated with mutants disrupted in the two genes, Mutants bearing single-site deletions in norB or norZ were affected n either in aerobic nor in anaerobic growth with nitrate or nitrite as t he terminal electron acceptor, Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions, Anaerobic growt h was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid, These results show that the norB and n orZ gene products are isofunctional and instrumental in denitrificatio n.