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

scholarly journals Type 2-depleted fungal laccase

1988 ◽  
Vol 253 (2) ◽  
pp. 561-568 ◽  
Author(s):  
P M Hanna ◽  
D R McMillin ◽  
M Pasenkiewicz-Gierula ◽  
W E Antholine ◽  
B Reinhammar
Keyword(s):  
Molar Absorptivity ◽  
Reduced Form ◽  
Intramolecular Electron Transfer ◽  
Visible Absorption ◽  
Fungal Laccase ◽  
Covalently Bonded ◽  
High Concentrations ◽  
Type 3

Although copper is quantitatively removed from fungal laccase (Polyporus versicolor) by extended dialysis against high concentrations of cyanide, we have been unable to reconstitute the protein by addition of Cu(I) ions. However, two new methods for reversibly removing the type 2 Cu centre have been developed. The visible absorption at 610 nm, which is attributable to type 1 Cu, is unaffected by the procedure, but the absorbance of the type 3 Cu at 330 nm is decreased by 60 +/- 10%. The decrease is due, at least in part, to partial reduction of the binuclear type 3 centre, although there may be some change in the molar absorptivity of the oxidized chromophore as well. The change in the c.d. spectrum that occurs at approx. 350 nm may be explained in the same way, but it may also reflect the loss of a signal due to the type 2 Cu. Upon removal of the type 2 Cu an absorbance increase appears at approx. 435 nm, and it is assigned to the semi-reduced form of the type 3 pair. In the e.p.r. spectrum of the type 2-depleted enzyme the type 1 Cu signal exhibits well-resolved ligand hyperfine splitting, which can be simulated on the basis of contributions from two N and two H nuclei (AH congruent to AN congruent to 25 MHz). The H atoms are assumed to be attached to the beta-carbon of the covalently bonded cysteine ligand. A signal from a semi-reduced form(s) of the type 3 site can also be resolved in the spectrum of the type 2-depleted enzyme, and on the basis of the second integral of the e.p.r. spectrum 40% of the type 3 pairs are believed to be in a partially reduced state. The semi-reduced type 3 site is remarkably stable and is not readily oxidized by H2O2 or IrCl6(2-) or reduced by Fe(CN)6(4-). Intramolecular electron transfer is apparently quite slow in at least some forms of the type 2-depleted enzyme, and this may explain why the activity is at best 5% of that of the native enzyme. Full activity returns when type 2 copper is restored.

scholarly journals Unmediated heterogeneous electron transfer reaction of ascorbate oxidase and laccase at a gold electrode

1998 ◽  
Vol 332 (3) ◽  
pp. 611-615 ◽  
Author(s):  
Roberto SANTUCCI ◽  
Tommaso FERRI ◽  
Laura MORPURGO ◽  
Isabella SAVINI ◽  
Luciana AVIGLIANO
Keyword(s):  
Electron Transfer ◽  
Electrode Surface ◽  
Gold Electrode ◽  
Ascorbate Oxidase ◽  
Cyclic Voltammograms ◽  
Intramolecular Electron Transfer ◽  
Redox Potentials ◽  
Catalytic Current ◽  
Heterogeneous Electron Transfer

The unmediated electrochemistry of two large Cu-containing proteins, ascorbate oxidase and laccase, was investigated by direct-current cyclic voltammetry. Rapid heterogeneous electron transfer was achieved in the absence of promoters or mediators by trapping a small amount of protein within a solid, electrochemically inert, tributylmethyl phosphonium chloride membrane coating a gold electrode. The problems typical of proteins in solution, such as adsorption on the electrode surface, were avoided by this procedure. In anaerobic conditions, the cyclic voltammograms, run at a scan rate of up to 200 mV/s, showed the electron transfer process to be quasi-reversible and diffusion-controlled. The pH-dependent redox potentials (+360 mV and +400 mV against a normal hydrogen electrode at pH 7.0 for ascorbate oxidase and laccase respectively and +390 mV and +410 mV at pH 5.5) were similar to those of the free proteins. The same electrochemical behaviour was recorded for the type 2 Cu-depleted derivatives, which contain reduced type 3 Cu, whereas the apoproteins were electrochemically inactive. Under aerobic conditions the catalytic current intensity of holoprotein voltammograms increased up to approx. 2-fold at a low scanning rate, with unchanged redox potentials. The voltammograms of type 2 Cu-depleted proteins and of apoproteins were unaffected by the presence of oxygen. This suggests that electron uptake at the electrode surface involves type 1 Cu and that only in the presence of oxygen is the intramolecular electron transfer to other protein sites rapid enough to be observed. The analogy with available kinetic results is discussed.

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.

Type 2 Diabetes Overtakes Type 1 in Hispanic Girls

2008 ◽  
Vol 38 (15) ◽  
pp. 18
Author(s):  
SHERRY BOSCHERT
Keyword(s):  
Type 2 Diabetes
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