6-S-cysteinylation of bi-covalently attached FAD in berberine bridge enzyme tunes the redox potential for optimal activity.
A mutagenic analysis of the amino acid residues His-104 and Cys-166, which are involved in the bi-covalent attachment of FAD to berberine bridge enzyme, was performed. Here we present a detailed biochemical characterization of the cysteine link to FAD observed in this recently discovered group of flavoproteins. The C166A mutant protein still has residual activity, but reduced to approximately 6% of the turnover rate observed for wild-type berberine bridge enzyme. A more detailed analysis of single reaction steps by stopped-flow spectrophotometry showed that the reductive half-reaction is greatly influenced by the lack of the 6-S-cysteinyl linkage, resulting in a 370-fold decrease in the rate of flavin reduction. Determination of the redox potentials for both wild type and the C166A mutein revealed that the difference in the redox potential observed can fully account for the change in the kinetic properties. The wild-type protein exhibits a midpoint potential of +132 mV, which is the highest redox potential determined for any flavoenzyme so far. Removal of the cysteine linkage to FAD in the C166A mutein leads to a redox potential of +53 mV, which is in the expected range for flavoproteins with a single covalent attachment of FAD to a His residue via its 8-alpha position. We also show that the biochemical properties of the mutein resemble that of typical flavoprotein oxidases and that deviations from this behavior observed for the wild type are due to the FAD-6-S-cysteinyl bond. In addition, rapid reaction stopped-flow experiments give no indication for a radical mechanism supporting the direct transfer of a hydride from the substrate to the cofactor.
Winkler
Andreas
A
Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
Kutchan
Toni M
TM
Macheroux
Peter
P
D003545:Cysteine / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins; D013057:Spectrum Analysis
D010940:Plant Proteins; D005182:Flavin-Adenine Dinucleotide; C071690:reticuline oxidase; D010089:Oxidoreductases, N-Demethylating; D003545:Cysteine