The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsulatus Xanthine Dehydrogenase

Abstract

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (␣␤) 2 heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (␣␤) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (␣␤) subunits has to precede Moco insertion. In an (␣␤) 2 XDH heterotetramer variant, including only one active Moco-center, the active (␣␤) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (␣␤) 2 heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (␣␤) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation. Xanthine oxidoreductases (XORs)2 are the best studied enzymes of the small but important class of molybdenum-containing iron-sulfur flavoproteins, containing two non-identical [2Fe2S] clusters, FAD, and the sulfurated form of the molybdenum cofactor (Moco) as catalytically acting units (1-3).Mammalian XORs catalyze the hydroxylation of hypoxanthine and xanthine, the last two steps in the formation of urate, and exist originally as the dehydrogenase form (XDH, EC 1.17.1.4) but can be converted to the oxidase form (XO, EC 1.1.3.22) either reversibly by oxidation of sulfhydryl residues of the protein molecule or irreversibly by proteolysis (4). XDH shows a preference for NAD ϩ reduction at the FAD reaction site, whereas XO exclusively uses dioxygen as a terminal electron acceptor, leading to the formation of superoxide and hydrogen peroxide (5). The enzyme has been implicated in diseases characterized by oxygen radical-induced tissue damage, such as postischemic reperfusion injury (6). The oxidation of xanthine takes place at the molybdenum center, and the electrons thus introduced are rapidly distributed to the other centers according to their relative redox potentials (1). The re-oxidation of the reduced enzyme by the oxidant substrate, either NAD ϩ or molecular oxygen, occurs through FAD (7). The two [2Fe2S] clusters (FeSI and FeSII) are indistinguishable in terms of their absorption spectra, but the midpoint redox potential of FeSII is generally more positive than that of the FeSI center (8, 9). The FeS centers from enzymes of the XO family have been characterized earlier by EPR (10 -12). The FeSI center of eukaryotic XOR exh...

Characterization and Crystallization of Mouse Aldehyde Oxidase 3: From Mouse Liver toEscherichia coliHeterologous Protein Expression

“…Both recombinant and native mAOX3 were compared in detail on the basis of their spectroscopic properties. The EPR spectra of mAOX3 were found to be very similar to those from the mAOX1 protein, showing a rhombic signal for FeSI (Schumann et al, 2008). There are essentially no differences in the g values and line widths for the native and the recombinant enzymes.…”

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

“…The arginine is located close to FeSI, and when this mutation was introduced in the Rhodobacter capsulatus xdhB gene and the corresponding RcXDH-R135C was characterized, the purified protein also existed in two forms, a monomeric inactive form and a dimeric active form (Leimkühler et al, 2003). Further analyses of the monomer/dimer behavior of R. capsulatus XDH resulted in a model in which it was proposed that dimerization requires that the two FeS clusters are assembled correctly before Moco can be inserted and the protein can dimerize via the Moco domain (Schumann et al, 2008). Thus, amino acid exchanges in proximity to the FeS clusters influence the structure of the protein in a manner that dimerization is no longer effective.…”

Xanthine dehydrogenase: An old enzyme with new knowledge and prospects

“…17,27 Leimkuhler group has proposed a pioneering mechanism for the assembly and cofactor insertion of R. capsulatus (ab) 2 XDH. 28 According to the mechanism, the biosynthesis of active XDH is proposed to involve 5 processes: 1) synthesis of apoproteins consisting of the 3 domains, depicted as S, M and L; 2) formation of heteromultimer apoprotein in the case of heteromultimer multi-subunit XDH; 3) insertion of 2 [2Fe-2S] clusters and a FAD into the S and M domains respectively; 4) dimerization of apoproteins assembled with [2Fe-2S] and FAD cofactors via the L domains; 5) insertion of sulfurated Moco into the L domains with the help of chaperone protein XdhC (Fig. 3 part II).…”