Genomic DNA Cloning of the Region Encoding Nitric Oxide Reductase in Paracoccus halodenitrificansand a Structure Model Relevant to Cytochrome Oxidase

Institute for Molecular Science, Okazaki National Research Institutes, Okazaki, Japan.
Biochemical and Biophysical Research Communications (Impact Factor: 2.41). 01/1998; 243(2):400-406. DOI: 10.1006/bbrc.1998.8106

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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Myoglobin (Mb) is an ideal scaffold protein for rational protein design mimicking native enzymes. We recently designed a nitrite reductase (NiR) based on sperm whale Mb by introducing an additional distal histidine (Leu29 to His29 mutation) and generating a distal tyrosine (Phe43 to Tyr43 mutation) in the heme pocket, namely L29H/F43Y Mb, to mimic the active site of cytochrome cd (1) NiR from Ps. aeruginosa that contains two distal histidines and one distal tyrosine. The molecular modeling and dynamics simulation study herein revealed that L29H/F43Y Mb has the necessary structural features of native cytochrome cd (1) NiR and can provide comparable interactions with nitrite as in native NiRs, which provides rationality for the protein design and guides the protein engineering. Additionally, the present study provides an insight into the relatively low NiR activity of Mb in biological systems.
    Journal of Molecular Modeling 05/2012; 18(9):4409-15. · 1.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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; · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe(B) site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by approximately 110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a approximately 100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe(B) site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.
    Proceedings of the National Academy of Sciences 05/2010; 107(19):8581-6. · 9.81 Impact Factor