Protein Film Voltammetry of Rhodobacter Capsulatus Xanthine Dehydrogenase

Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes the hydroxylation of xanthine to uric acid with NAD⁺ as the electron acceptor. R. capsulatus XDH forms an (αβ)₂ heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures of bovine XDH and R. capsulatus XDH showed that the two proteins have highly similar folds; however, R. capsulatus XDH is at least 5 times more active than bovine XDH and, unlike mammalian XDH, does not undergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinant enzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite working electrode. Furthermore, we have determined all redox potentials of the four cofactors (Mo^VI/V, Mo^V/IV, FAD/FADH, FADH/FADH₂ and two distinct [2Fe-2S]^2+/+ clusters) using a combination of potentiometric and voltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potential for the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis.