Keggin-type polyoxotungstates as mushroom tyrosinase inhibitors - A speciation study

Mushroom tyrosinase abPPO4 is a commercially relevant polyphenol oxidase and has been being targeted for numerous inhibition studies including polyoxometalates (POMs). In the present work, its diphenolase activity was inhibited at pH 6.8 by a series of structurally related polyoxotungstates (POTs) of the α-Keggin archetype, exhibiting the general formula [Xⁿ⁺W₁₂O₄₀]^(8-n)- in order to elucidate charge-dependent activity correlations. Kinetic data were obtained from the dopachrome assay and ¹⁸³W NMR was applied to obtain crucial insights into the actual Keggin POT speciation in solution, facilitating a straightforward assignment of inhibition effects to the identified POT species. While [PW₁₂O₄₀]^3- was completely hydrolyzed to its moderately active lacunary form H_x[PW₁₁O₃₉]^(7-x)- (K_i = 25.6 mM), [SiW₁₂O₄₀]^4- showed the most pronounced inhibition effects with a K_i of 4.7 mM despite of partial hydrolysis to its ineffective lacunary form H_x[SiW₁₁O₃₉]^(8-x)-. More negative Keggin cluster charges of 5- and 6- generally resulted in preclusion of inhibitory efficacy as well as hydrolysis, but with the Ni-substituted cluster [PW₁₁O₃₉{Ni(H₂O)}]^5- enzymatic inhibition was clearly restored (K_i = 9.7 mM). The inhibitory capacity of the structurally intact Keggin POTs was found to be inversely correlated to their net charge. The here applied speciation strategy is of utmost importance for any biological POM application to identify the actually active POM species.