Properties of the Trp290His variant of Fusarium NRRL 2903 galactose oxidase: interactions of the GOase_semi state with different buffers, its redox activity and ability to bind azide

Abstract

 The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short ∼8 Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400 mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25  °C, I=0.100 M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the non-radical GOase_semi (Cu^II) form of the Trp290His variant. Equilibrium constants K _eq/M^–1 from absorbance changes at 635 nm are for 1 : 1 interactions with the OH-containing buffers H₂PO₄ ^– (231), Hepes (43) and Tris (202), concentrations 0–60 mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at ∼635 nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOase_ox/GOase_semi couple is 730 mV, which compares with 400 mV for the WT GOase couple. Consistent with the 730 mV value the GOase_semi form is not oxidised with [Fe(CN)₆]^3– (410 mV) or [W(CN)₈]^3– (530 mV), and much stronger oxidants such as [Mo(CN)₈]^3– (800 mV) and [IrCl₆]^2– (890 mV) are required. The GOase_ox product is unstable and decays within 20 min with re-formation of GOase_semi. From changes in UV-Vis spectra with pH, Trp290His GOase_semi gives a pK _a of 6.9, and rate constants for the oxidation of GOase_semi with [Mo(CN)₈]^3– are dependent on this same pK _a. The latter compares with 7.9 for WT GOase_semi, and is assigned here also as protonation of Tyr-495. The 1 : 1 binding of azide at the substrate-binding (H₂O) site of Trp290His GOase_semi was studied and gives a formation constant 330 M^–1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOase_semi. The trends observed indicate less affinity of Trp290His GOase_semi for the ionic reactants H⁺ and N₃ ^–.