EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the type 2 copper centers of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015)

Biochemistry ◽  
1994 ◽  
Vol 33 (11) ◽  
pp. 3171-3177 ◽  
Author(s):  
Barry D. Howes ◽  
Zelda H. L. Abraham ◽  
David J. Lowe ◽  
Thomas Bruser ◽  
Robert R. Eady ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Double Resonance ◽  
Electron Nuclear Double Resonance ◽  
Dissimilatory Nitrite Reductase

scholarly journals Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

2001 ◽  
Vol 183 (14) ◽  
pp. 4149-4156 ◽  
Author(s):  
Hirotaka Ichiki ◽  
Yoko Tanaka ◽  
Kiyotaka Mochizuki ◽  
Katsuhiko Yoshimatsu ◽  
Takeshi Sakurai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Nitrite Reductase ◽  
Sodium Dodecyl ◽  
Resonance Spectroscopy ◽  
Halophilic Archaeon ◽  
Haloarcula Marismortui ◽  
Dissimilatory Nitrite Reductase

ABSTRACT Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (gII = 2.232; AII = 4.4 mT) and type 2 Cu centers (gII = 2.304; AII = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 μM NO2 − · min−1 · mg−1.

Dissimilatory nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) is trimeric and contains both type 1 and type 2 copper centres.

1993 ◽  
Vol 51 (1-2) ◽  
pp. 359
Author(s):  
Z. Abraham ◽  
E.T. Adman ◽  
T. Brüser ◽  
R.R. Eady ◽  
J.G. Grossmann ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Dissimilatory Nitrite Reductase

A continuous-wave electron–nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine β-mono-oxygenase of the adrenal medulla

2002 ◽  
Vol 363 (3) ◽  
pp. 677-686
Author(s):  
Bettina KATTERLE ◽  
Rudolf I. GVOZDEV ◽  
Ntei ABUDU ◽  
Torbjørn LJONES ◽  
K. Kristoffer ANDERSSON
Keyword(s):  
Continuous Wave ◽  
Dipolar Coupling ◽  
Double Resonance ◽  
Coupling Constants ◽  
Methylococcus Capsulatus ◽  
Electron Nuclear Double Resonance ◽  
Strongly Coupled ◽  
Copper Site ◽  
Typical Type

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu2+ centre with the following EPR parameters: gz 2.24, gx,y 2.06, ACuz 19.0mT and ACux,y 1.0mT. Simulation of the Cu2+ spectrum yielded a best match by using four equivalent nitrogens (AN = 1.5mT, 42MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu2+, in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron—nuclear double resonance (CW ENDOR; 9.4GHz, 5–20K) experiments at g⊥ of the Cu(II) spectrum show a weak coupling to protons with an AH of 2.9MHz that corresponds to a distance of 3.8Å (1Å≡0.1nm), assuming that it is a purely dipolar coupling. Incubation in 2H2O leads to a significant decrease in these 1H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu2+ by diethyldithiocarbamic acid. The 1H and 14N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu2+ enzyme, dopamine β-mono-oxygenase (DβM). For DβM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and 2H2O-exchangeable protons (2.9MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40MHz) from the co-ordinating N of the histidines in DβM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

scholarly journals Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres

1993 ◽  
Vol 295 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Z H L Abraham ◽  
D J Lowe ◽  
B E Smith
Keyword(s):  
Nitrite Reductase ◽  
Specific Activity ◽  
Visible Region ◽  
Exchange Chromatography ◽  
Published Data ◽  
Absorption Bands ◽  
Sds Page ◽  
Dissimilatory Nitrite Reductase

Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme had a subunit M(r) of 36.5 kDa. The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500. E.p.r. spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data [Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J. Biochem. (Tokyo) 96, 447-454], where only type 1 copper centres were reported. Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously. As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm. The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases. We conclude that, in contrast with previous reports, this ‘blue’ nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.

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