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: 2.3). 02/1998; 243(2):400-406. DOI: 10.1006/bbrc.1998.8106


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: 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 · 2.30 Impact Factor
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    Biochimica et Biophysica Acta 06/1999; 1411(2-3):456-74. DOI:10.1016/S0005-2728(99)00032-8 · 4.66 Impact Factor
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    ABSTRACT: Hydrogenovibrio marinus strain MH-110, an obligately lithoautotrophic hydrogen-oxidizing bacterium, possesses three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO): namely, two form I type (cbbLS-1 and cbbLS-2) and one form II type (cbbM) enzymes. The cbbQ-m gene was located downstream of cbbM, and showed high similarity to other cbbQ genes and nirQ/norQ genes in denitrifying bacteria. Phylogenetic analysis of CbbQ and NirQ/NorQ indicated that CbbQ-m from Hv. marinus closely resembled CbbQ from Thiobacillus intermedius and Thiobacillus neapolitannus and less closely resembled NirQ and NorQ. The cbbQ-m gene has been shown to activate the form II RubisCO in E. coli cells, and the cbbQ-t from Hydrogenophilus thermoluteolus could also activate the form II RubisCO. Both cbbQ genes have also been shown to activate the form I RubisCO from Hp. thermoluteolus in E. coli cells. However, the activation levels of two form I RubisCOs from Hv. marinus were smaller than that of form I RubisCOs from Hp. thermoluteolus. Form II RubisCO activated by CbbQ-m (QM) was purified from E. coli cells. The result of the 8-anilino-1-naphthalenesulfonate binding assay and the circular dichroism spectra indicated that QM was conformationally different from Form II RubisCO that was not activated by CbbQ.
    Biochemical and Biophysical Research Communications 12/1999; 265(1):177-83. DOI:10.1006/bbrc.1999.1103 · 2.30 Impact Factor
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