On the iron–sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase
Open Access
- 1 June 2000
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 267 (12), 3640-3646
- https://doi.org/10.1046/j.1432-1327.2000.01393.x
Abstract
Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron–sulfur flavoenzyme that catalyses the first and rate‐limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid‐labile sulfur, which are most likely arranged into four [4Fe−4S] clusters. However, the presence and role of such Fe–S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox‐inactive during absorbance‐monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe−4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR‐monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR‐active species was present in the enzyme ‘as isolated’. In full agreement with absorbance‐monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe−4S]2+,1+ clusters, which both exhibit a midpoint potential of ≈ −0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half‐field signal around g≈ 3.9. Under no circumstances could the other two postulated Fe–S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe−4S] clusters, presumably determined by the C‐terminal eight‐iron ferredoxin‐like module of the protein, whose participation in the enzyme‐catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe−4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N‐terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase β subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe−4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N‐terminus of the glutamate synthase β subunit, which are conserved in dihydropyrimidine dehydrogenase and in several β‐subunit‐like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe−4S] clusters of low to very low midpoint potential.Keywords
This publication has 30 references indexed in Scilit:
- Porcine Recombinant Dihydropyrimidine Dehydrogenase: Comparison of the Spectroscopic and Catalytic Properties of the Wild-Type and C671A Mutant EnzymesBiochemistry, 1998
- Mossbauer Study of 4-Hydroxybutyryl-CoA Dehydratase Probing the Role of an Iron-Sulfur Cluster in an Overall Non-Redox ReactionEuropean Journal of Biochemistry, 1997
- Properties of the Recombinant β Subunit of Glutamate SynthaseEuropean Journal of Biochemistry, 1996
- 4-Hydroxybutyryl-CoA Dehydratase from Clostridium aminobutyricum: Characterization of FAD and Iron−Sulfur Clusters Involved in an Overall Non-Redox ReactionBiochemistry, 1996
- Reversible Super‐Reduction of the Cubane [4Fe‐4S](3+;2+;1+) in the High‐Potential Iron‐Sulfur Protein Under Non‐Denaturing ConditionsEuropean Journal of Biochemistry, 1995
- Electrochemical study of the redox properties of [2Fe‐2S] ferredoxins Evidence for superreduction of the Rieske [2Fe‐2S] clusterFEBS Letters, 1995
- Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopiesBiochemistry, 1992
- Purification and characterization of dihydropyrimidine dehydrogenase from pig liverEuropean Journal of Biochemistry, 1989
- Quantitative numerical analysis of g strain in the EPR of distributed systems and its importance for multicenter metalloproteinsJournal of Magnetic Resonance (1969), 1985
- The Pyruvate Dehydrogenase Complex of Escherichia coli K12European Journal of Biochemistry, 1983