Protein Film Voltammetry of Rhodobacter Capsulatus Xanthine Dehydrogenase
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- 作者：Kondo Franç ; ois Aguey-Zinsou ; Paul V. Bernhardt ; and Silke Leimkü ; hler
- 刊名：Journal of the American Chemical Society
- 出版年：2003
- 出版时间：December 17, 2003
- 年：2003
- 卷：125
- 期：50
- 页码：15352 - 15358
- 全文大小：144K
- 年卷期：v.125,no.50(December 17, 2003)
- ISSN：1520-5126
文摘
Xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus catalyzes thehydroxylation of xanthine to uric acid with NAD+ as the electron acceptor. R. capsulatus XDH forms an(
)2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures ofbovine 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 notundergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinantenzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite workingelectrode. Furthermore, we have determined all redox potentials of the four cofactors (MoVI/V, MoV/IV, FAD/FADH, FADH/FADH2 and two distinct [2Fe-2S]2+/+ clusters) using a combination of potentiometric andvoltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potentialfor the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highestpotential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switchduring voltammetry that precedes catalysis.
)2 heterotetramer and is highly homologous to homodimeric eukaryotic XDHs. The crystal structures ofbovine 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 notundergo the conversion to the oxidase form. Here we demonstrate electrocatalytic activity of the recombinantenzyme, expressed in Escherichia coli, while immobilized on an edge plane pyrolytic graphite workingelectrode. Furthermore, we have determined all redox potentials of the four cofactors (MoVI/V, MoV/IV, FAD/FADH, FADH/FADH2 and two distinct [2Fe-2S]2+/+ clusters) using a combination of potentiometric andvoltammetric methods. A novel feature identified in catalytic voltammetry of XDH concerns the potentialfor the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highestpotential cofactor. This unusual observation is explained on the basis of a pterin-associated oxidative switchduring voltammetry that precedes catalysis.