Kinetic Studies on the Redox Interconversion of GOase_semiand GOase_oxForms of Galactose Oxidase with Inorganic Complexes as Redox Partners

Abstract

Redox interconversions between the GOase(semi) (Cu(II), Tyr) and tyrosyl radical containing GOase(ox) (Cu(II), Tyr(*)) oxidation states of the Cu-containing enzyme galactose oxidase (GOase) from Fusarium NRRL 2903 have been studied. The inorganic complexes [Fe(CN)(6)](3)(-) (410 mV), [Co(phen)(3)](3+) (370 mV), [W(CN)(8)](3)(-) (530 mV), and [Co(dipic)(2)](-) (362 mV) (E degrees ' values vs NHE; dipic = 2,6-dicarboxylatopyridine) were used as oxidants for GOase(semi), and [Fe(CN)(6)](4)(-) and [Co(phen)(3)](2+) as reductants for GOase(ox). On oxidation of GOase(semi) a radical is generated at the coordinated phenolate of Tyr-272 to give GOase(ox). The one-electron reduction potential E degrees ' (25 degrees C) for the GOase(ox)/GOase(semi) couple varies with pH and is 400 mV vs NHE at pH 7.5, the smallest value so far observed for a tyrosyl radical. The reactions are very sensitive to pH, or more precisely to pK(a) values of GOase(semi) and GOase(ox), and the charge on the inorganic reagent. For example, with [Fe(CN)(6)](3)(-) as oxidant, the rate constant (25 degrees C)/M(-)(1) s(-)(1) of 0.16 x 10(3) (pH approximately 9.5) increases to 4.3 x 10(3) (pH approximately 5.5), while for [Co(phen)(3)](3+) a value of 4.9 x 10(3) (pH approximately 9.5) decreases to 0.04 x 10(3) (pH approximately 5.5), I = 0.100 M (NaCl). From the kinetics a single GOase(semi) acid dissociation process, pK(a) = 8.0 (average), has been confirmed by UV-vis spectrophotometric studies (7.9). The corresponding value for GOase(ox) is 6.7. No comparable kinetic or spectrophotometric pH dependences are observed with the Tyr495Phe variant, indicating the axial Tyr-495 as the site of protonation. Neutral CH(3)CO(2)H and HN(3) species bind at the substrate binding site of GOase(semi), thus mimicking the behavior of primary alcohols RCH(2)OH, the natural substrate of GOase. On coordination, loss of a proton occurs, and inhibition of the oxidation with [Fe(CN)(6)](3)(-) is observed.

Cobalt(II) and Cobalt(III) Dipicolinate Complexes: Solid State, Solution, and in Vivo Insulin-like Properties

“…Normal untreated animals were matched in age and weight. Treatment consisted of daily adding VOSO 4 (0.78 mg/mL (3.1 mM) for days 1-14) or [Co(dipic) 2 ] 2-(0.50 mg/mL (0.84 mM) for days 1-3 and 0.60 mg/mL (1.01 mM) for days [4][5][6][7][8][9][10][11][12][13][14]. The amounts of compound were calculated including 7 water hydrates for K 2 [Co-(dipic) 2 ] and 5 for VOSO 4 .…”

Copper(II)‐Coordinated α‐Azophenols: Effect of the Metal‐Ion Geometry on Phenoxyl/Phenolate Oxidation Potential and Reactivity

“…The redox potentials for the interconversion between these three forms were shown to be pH dependent. The Cu II /Cu I redox potential ranges between 0.16 (pH 5.5) and 0.12 V (pH 10.5), with a value of 0.15 V versus the normal hydrogen electrode (NHE) at pH 7.5,3 whereas the Y 272 · /Y 272 redox potential ranges between 0.50 (pH 5.5) and 0.38 V (pH 8.5), with a value of 0.40 V vs. NHE at pH 7.5 4. This pH dependency is ascribed to the protonation equilibrium of the axial tyrosine Y 495 , which becomes a weaker donor at low pH values.…”

Die Koordination von Methoxid in der Tasche von {CuIITpCum,Me} und ein einfaches Modell für das Cu-Zentrum der Galactose-Oxidase

“…[4] Ein ungewöhnliches Cysteinyltyrosin (Tyr272, Cys228) ist über das Tyrosin-O-Atom an der Pyramidenbasis an Cu koordiniert und erweitert dessen Redoxaktivität. [5] Zwei weitere basale Koordinationsstellen sind von Histidin-N-Atomen besetzt, die apicale Stelle von einem O-Atom eines normalen Tyrosins (Tyr495). Die vierte Koordinationsstelle an der Pyramidenbasis direkt neben dem Tyr272-O-Atom ist die Substratbindungsstelle.…”

“…[6] Die Oxidation von GOase SEMI zur aktiven GOase OX findet am Cysteinyltyrosin statt. Sykes et al bestimmten das Oxidationspotential dieses Tyrosins und stellten einen ungewöhnlich niedrigen Wert von 0.40 V (gegen die Normalwasserstoffelektrode, pH 7.5) fest, [5] während Tyrosin und Phenolate sonst üblicherweise bei Potentialen oxidiert werden, die näher bei 1.0 V liegen. [9] Mit der Substratoxidation geht die Reduktion von molekularem Sauerstoff einher [Gl.…”

“…[7, 13±15] Er ist auch trotz struktureller ¾hnlichkeit zwischen dem Komplex 2 und GOase OX um etwa 0.6 V positiver als das Reduktionspotential des Tyrosylrestes in der GOase OX . [5] Ein Charakteristikum im Enzym, das zum negativeren Wert für Tyr272 beitragen könnte, mag die Bildung eines Charge-Transfer-Komplexes mit dem Indolring von Trp290 sein. [6] Der Cumenylsubstituent des apicalen Pyrazols in 2 könnte eine ähnliche p-Stapelung mit dem Phenolatring eingehen.…”