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Reduction of NO to N2O by denitrifiying bacteria is catalyzed either by a monomeric quinol-nitric oxide reductase (qNor) or by a heterodimeric cytochrome c-dependent nitric oxide reductase (cNor). In ancient thermophilic bacteria belonging to the Thermales and Aquificales phylogenetic groups, the cluster encoding the cNor includes a small third gene (norH), in addition to those encoding homologues to the subunits of a typical cNor (norC and norB). We show in Thermus thermophilus that the three genes are cotranscribed in a single mRNA from an inducible promoter. The isolation of individual nor mutants and the production in vivo of His-tagged NorH protein followed by immobilized-metal affinity chromatography (IMAC) allowed us to conclude that NorH constitutes a third subunit of the cNor from T. thermophilus, which is involved in denitrification in vivo, likely allowing more efficient electron transport to cNor., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471-480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca(2+), a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR.