Recombinant Rhodobacter capsulatus xanthine dehydrogenase, a useful model system for the characterization of protein variants leading to xanthinuria I in humans.

[]

Rhodobacter capsulatus xanthine dehydrogenase (XDH ) forms an (alphabeta )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 . We have developed an efficient system for the recombinant expression of R . capsulatus XDH in Escherichia coli . The recombinant protein shows spectral features and a range of substrate specificities similar to bovine milk xanthine oxidase . 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 . EPR spectra were obtained for the FeS centers of the enzyme showing an axial signal for FeSI , which is different from that reported for xanthine oxidase . X-ray absorption spectroscopy at the iron and molybdenum K-edge and the tungsten LIII-edge have been used to probe the different metal coordinations of variant forms of the enzyme . Based on a mutation identified in a patient suffering from xanthinuria I , the corresponding arginine 135 was substituted to a cysteine in R . capsulatus XDH , and the protein variant was purified and characterized . Two different forms of XDH -R 135 C were purified , an active (alphabeta )2 heterotetrameric form and an inactive (alphabeta ) heterodimeric form . The active form contains a full complement of redox centers , whereas in the inactive form the FeSI center is likely to be missing .