Catalytic Function and Local Proton Structure at the Type 2 Copper of Nitrite Reductase:  The Correlation of Enzymatic pH Dependence, Conserved Residues, and Proton Hyperfine Structure†

Biochemistry ◽  
2002 ◽  
Vol 41 (23) ◽  
pp. 7464-7474 ◽  
Author(s):  
Yiwei Zhao ◽  
Dmitriy A. Lukoyanov ◽  
Yuriy V. Toropov ◽  
Kenneth Wu ◽  
James P. Shapleigh ◽  
...  
Keyword(s):  
Hyperfine Structure ◽  
Nitrite Reductase ◽  
Ph Dependence ◽  
Conserved Residues ◽  
Proton Structure ◽  
Catalytic Function

scholarly journals pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans

1997 ◽  
Vol 324 (2) ◽  
pp. 511-516 ◽  
Author(s):  
Zelda. H. L ABRAHAM ◽  
Barry. E SMITH ◽  
Barry. D HOWES ◽  
David. J LOWE ◽  
Robert. R EADY
Keyword(s):  
Nitrite Reductase ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Binding Constants ◽  
Single Type ◽  
Nitrite Ion ◽  
Cu Content ◽  
Steady State Kinetics ◽  
Decreasing Ph

The first quantitative characterization of the interaction of NO2- with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidansusing steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface. Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8. Equilibrium gel filtration showed that binding of NO2- to the oxidized NiR was also pH-dependent. At pH 7.5, no binding was detectable, but binding was detectable at lower pH values. At pH 5.2, the concentration-dependence for binding of NO2- to the enzyme showed that approx. 4.1 NO2- ions bound per trimeric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO2- ions per trimer. When corrected for this binding, a value of 3 NO2- ions bound per trimer of NiR, equivalent to the type-2 Cu content. The NO2--induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity. Binding constants for NO2- at a single type of site, after allowing for the non-specifically bound NO2-, were 350±35 μM (mean±S.E.M.) at pH 7.5 and <30 μM at pH 5.2. The apparent Km for NO2- with saturating concentrations of dithionite as reductant was 35 μM at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO2- binds.

scholarly journals The Blue Copper-Containing Nitrite Reductase fromAlcaligenes xylosoxidans: Cloning of the nirAGene and Characterization of the Recombinant Enzyme

1999 ◽  
Vol 181 (8) ◽  
pp. 2323-2329 ◽  
Author(s):  
Miguel Prudêncio ◽  
Robert R. Eady ◽  
Gary Sawers
Keyword(s):  
Amino Acid ◽  
Nitrite Reductase ◽  
Recombinant Enzyme ◽  
Leader Peptide ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Blue Copper

ABSTRACT The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sources. The full-length protein included a 24-amino-acid leader peptide. The nirA gene was overexpressed inEscherichia coli and was shown to be exported to the periplasm. Purification was achieved in a single step, and analysis of the recombinant Nir enzyme revealed that cleavage of the signal peptide occurred at a position identical to that for the native enzyme isolated from A. xylosoxidans. The recombinant Nir isolated directly was blue and trimeric and, on the basis of electron paramagnetic resonance spectroscopy and metal analysis, possessed only type 1 copper centers. This type 2-depleted enzyme preparation also had a low nitrite reductase enzyme activity. Incubation of the periplasmic fraction with copper sulfate prior to purification resulted in the isolation of an enzyme with a full complement of type 1 and type 2 copper centers and a high specific activity. The kinetic properties of the recombinant enzyme were indistinguishable from those of the native nitrite reductase isolated from A. xylosoxidans. This rapid isolation procedure will greatly facilitate genetic and biochemical characterization of both wild-type and mutant derivatives of this protein.

Effect of Arginine at Type 2 Cu Site on Spectroscopic Features and Enzymatic Activity of Copper-containing Nitrite Reductase

2007 ◽  
Vol 36 (1) ◽  
pp. 140-141
Author(s):  
Kazuya Yamaguchi ◽  
Aiko Tsuda ◽  
Masaki Nojiri ◽  
Shinnichiro Suzuki
Keyword(s):  
Enzymatic Activity ◽  
Nitrite Reductase

Characterization of Azide-Binding Type 2 Cu(II) Site of Nitrite Reductase

2001 ◽  
Vol 74 (1) ◽  
pp. 183-184
Author(s):  
Kunishige Kataoka ◽  
Shinnichiro Suzuki
Keyword(s):  
Nitrite Reductase ◽  
Azide Binding ◽  
Binding Type

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.

Sign in / Sign up

Export Citation Format

Share Document