Pulse Radiolysis Studies on Nitrite Reductase from Achromobacter cycloclastes IAM 1013: Evidence for Intramolecular Electron Transfer from Type 1 Cu to Type 2 Cu

1994 ◽  
Vol 116 (24) ◽  
pp. 11145-11146 ◽  
Author(s):  
Shinnichiro Suzuki ◽  
Takamitsu Kohzuma ◽  
Deligeer ◽  
Kazuya Yamaguchi ◽  
Nobuhumi Nakamura ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Nitrite Reductase ◽  
Pulse Radiolysis ◽  
Intramolecular Electron Transfer ◽  
Achromobacter Cycloclastes

The Intramolecular Electron Transfer between the Type 1 Cu and the Type 2 Cu in a Mutant ofHyphomicrobiumNitrite Reductase

2005 ◽  
Vol 34 (1) ◽  
pp. 36-37 ◽  
Author(s):  
Shinnichiro Suzuki ◽  
Takehiko Maetani ◽  
Kazuya Yamaguchi ◽  
Kazuo Kobayashi ◽  
Seiichi Tagawa
Keyword(s):  
Electron Transfer ◽  
Intramolecular Electron Transfer

Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction

2001 ◽  
Vol 353 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Miguel PRUDÊNCIO ◽  
Robert R. EADY ◽  
Gary SAWERS
Keyword(s):  
Electron Transfer ◽  
Enzyme Activity ◽  
Nitrite Reductase ◽  
Methyl Viologen ◽  
Spectroscopic Properties ◽  
Sodium Dithionite ◽  
Epr Spectrum ◽  
Characteristic Type

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp92 [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369Ő382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g‖ = 2.12 (A‖ = 0mT) and g⊥ = 2.30 (A⊥ = 6.4mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp92 → Asn (D92N) mutant showed no characteristic type 2hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp92 → Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.

scholarly journals Pulse-radiolysis studies on the interaction of one-electron-reduced species with ascorbate oxidase in aqueous solution

1983 ◽  
Vol 209 (1) ◽  
pp. 167-174 ◽  
Author(s):  
P O'Neill ◽  
E M Fielden ◽  
A Finazzi-Agrò ◽  
L Avigliano
Keyword(s):  
Aqueous Solution ◽  
Pulse Radiolysis ◽  
Ascorbate Oxidase ◽  
Intramolecular Electron Transfer ◽  
Protein Residues ◽  
Type 1 Copper ◽  
Type 3 ◽  
Reducing Equivalents

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.

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.

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