Genomic DNA Cloning of the Region Encoding Nitric Oxide Reductase in Paracoccus halodenitrificansand a Structure Model Relevant to Cytochrome Oxidase
ABSTRACT The structural genes for the NO reductase inParacoccus halodenitrificans,norC,norB, andnorQwere sequenced. ThenorCandnorBencode the cytochromec(NorC) and cytochromeb(NorB) subunits, respectively. The matured NorC (17258 Da, 148 residues) has a binding motif (CXYCH) for hemec, which is axially coordinated by His65 and Met115. NorB (52337 Da, 451 residues) has twelve putative transmembrane helices and the 19% sequence homology with the subunit I of cytochrome oxidase fromParacoccus denitrificans.Several histidine and glutamate residues were identified as the ligands for two hemesband a non-heme iron in comparison with the sequence of cytochrome oxidase. The higher-order model structures constructed from the amino acid sequences of NorC and NorB showed the topology of the helical segments and the locations of the metal centers.
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ABSTRACT: 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.Proceedings of the National Academy of Sciences 07/2013; 1817. DOI:10.1073/pnas.1301731110 · 9.81 Impact Factor
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ABSTRACT: Reactions ofParacoccus halodenitrificansnitric oxide reductase (NOR) containing four iron centers, a low spin hemec,a low spin hemeb,a high spin hemeband a non-heme iron, have been studied to show the roles of each iron center. Soon after reacting the resting (oxidized) NOR with L-ascorbate, the low spin hemecand low spin hemebwere reduced to a considerable extent but the high spin hemebwas still in the oxidized form and was reduced slowly. When CO acted on the reduced NOR, the high spin hemebcenter changed to a low spin state. On the other hand, when NO acted on the resting NOR, no apparent spectral change was observed. However, when NO acted on the reduced NOR (a steady state condition, excess dithionite is present), both of the low spin centers changed to be partly in the oxidized form. A small but clear new EPR signal with g = 4.1 appeared together with some new signals at the g = 2 region soon after the action of NO on the reduced NOR. During incubation at room temperature the nitrosyl-heme signal typical of 5-coordination developed. These results suggested that both the high spin-hemebcenter and the non-heme iron are the reaction centers and their reductions are indispensable for the enzyme process in contrast to the reaction mechanism proposed for the P-450 type NOR(P-450nor).Biochemical and Biophysical Research Communications 10/1998; 251(1):248-251. DOI:10.1006/bbrc.1998.9451 · 2.28 Impact Factor