Flavin-N5OOH Functions as both a Powerful Nucleophile and a Base in the Superfamily of Flavoenzymes.

Flavoenzymes can mediate a large variety of oxidation reactions through the activation of oxygen. However, the O2 activation chemistry of flavin enzymes is not yet fully exploited. Normally, the O2 activation occurs at the C4a site of the flavin cofactor, yielding the flavin C4a-(hydro)hydroperoxyl species in monooxygenases or oxidases. Using extensive MD simulations, QM/MM calculations and QM calculations, our studies reveal the formation of the common nucleophilic species, Flavin-N5OOH, in two distinct flavoenzymes (RutA and EncM). Our studies show that Flavin-N5OOH acts as a powerful nucleophile that promotes C-N cleavage of uracil in RutA, and a powerful base in the deprotonation of substrates in EncM. We reason that Flavin-N5OOH can be a common reactive species in the superfamily of flavoenzymes, which accomplish generally selective general base catalysis and C-X (X=N, S, Cl, O) cleavage reactions that are otherwise challenging with solvated hydroxide ion base. These results expand our understanding of the chemistry and catalysis of flavoenzymes.

Dynamic association of flavin cofactors to regulate flavoprotein function.

Flavoproteins are key players in numerous redox pathways in cells. Flavin cofactors FMN and FAD confer the required chemical reactivity to flavoenzymes. In most cases, the interaction between the proteins and the flavins is noncovalent, yet stronger in comparison to other redox-active cofactors, such as NADH and NADPH. The association is considered static, but this view has started to change with the recent discovery of the dynamic association of flavins and flavoenzymes. Six cases from different organisms and various metabolic pathways are discussed here. The available mechanistic details span the range from rudimentary, as in the case of the ER-resident oxidoreductase Ero1, to comprehensive, as for the bacterial respiratory complex I. The same holds true in regard to the assumed functional role of the dynamic association presented here. More work is needed to clarify the structural and functional determinants of the known examples. Identification of new cases will help to appreciate the generality of the new principle of intracellular flavoenzyme regulation.

Structure-Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

Redox Titration of Flavoproteins: An Overview.

Redox titration of flavoproteins allows to detect and analyze (1) the determinants of the stabilization of individual redox forms of the flavin by the protein; (2) the binding of the redox-active cofactor to the protein; (3) the effects of other components of the systems (such as micro- or macromolecular interactors) on parameters 1 and 2; (4) the pattern of electron flow to and from the flavin cofactor to other redox-active chemical species, including those present in the protein itself or in its physiological partners. This overview presents and discusses the fundamentals of the methodological approaches most commonly used for these purposes, and illustrates how data may be obtained in a reliable way, and how they can be read and interpreted.