Characterization of the nitric oxide reductase from Thermus thermophilus

Abstract

Nitrous oxide (N 2 O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N 2 O is incomplete biological dentrification, and the enzymes responsible for the release of N 2 O are NO reductases. It was recently reported that ambient emissions of N 2 O 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 Fe B . 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 Fe B 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.

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

“…Lys-53 C is present in T. thermophilus cNOR, but its role was not investigated. The reason for the differences between the results of our study and the study with T. thermophilus cNOR (18) is not known. It is possible that PW 1 is used in the P. denitrificans cNOR (and P. aeruginosa cNOR) but not in T. thermophilus cNOR.…”

“…We note that the Lys-54 C is actually more conserved than the Glu-58 C (100% compared with 77%; Table 1 and Ref. 18). Although we do not have an explanation for this at the moment, we do note that the sequences that do not have Glu-58 C cluster together in the alignment and belong to different subgroups of the cNOR family than the subgroup of P. aeruginosa and P. denitrificans (18).…”

“…1 and 5) and that the other suggested pathways cannot take over the role of pathway 1. We note that a different study, where several residues were mutated in the cNOR from Thermus thermophilus (18), reached the conclusion that there is no preferred pathway in (this) cNOR. They did not investigate PW 3, but they observed effects on turnover rates when changing the residue at the entrance of PW 2, D209E/N/L C , which is an alanine in P. denitrificans (Ala-149 C ) and a glutamate in P. aeruginosa (Glu-145 C ).…”

“…18). Although we do not have an explanation for this at the moment, we do note that the sequences that do not have Glu-58 C cluster together in the alignment and belong to different subgroups of the cNOR family than the subgroup of P. aeruginosa and P. denitrificans (18). The replacing residues are hydrophilic (Gln, His, or Asp), and there are charged residues in the flanking sequence (3 or less residues away) that might take over the role of the Glu-58 C in these cNORs.…”

Transferable Denitrification Capability of Thermus thermophilus

“…The three-dimensional (3D) structures of both the soluble domain and all of complex I of T. thermophilus have been recently obtained by X-ray crystallography (33,35). A putative type II NADH oxidase (36) and the tricarboxylic acid cycle component succinate dehydrogenase (complex II) provide electrons to the respiratory chain as well (37). Electrons are transferred to menaquinone-8, the major quinone in the membrane of this organism (38).…”