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

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 similar to 8 Angstrom access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr272, reduction potential 400 mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25 degrees 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 nonradical GOase(semi) (CUII) 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 similar to 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 N-3(-).