On the Molecular and Submolecular Structure of the Semiquinone Cations of Alloxazines and Isoalloxazines as Revealed by Electron-Paramagnetic-Resonance Spectroscopy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66144/1/j.1432-1033.1981.tb05295.x.pd

Understanding flavin electronic structure and spectra

Flavins have emerged as central to electron bifurcation, signaling, and countless enzymatic reactions. In bifurcation, two electrons acquired as a pair are separated in coupled transfers wherein the energy of both is concentrated on one of the two. This enables organisms to drive demanding reactions based on abundant low-grade chemical fuel. To enable incorporation of this and other flavin capabilities into designed materials and devices, it is essential to understand fundamental principles of flavin electronic structure that make flavins so reactive and tunable by interactions with protein. Emerging computational tools can now replicate spectra of flavins and are gaining capacity to explain reactivity at atomistic resolution, based on electronic structures. Such fundamental understanding can moreover be transferrable to other chemical systems. A variety of computational innovations have been critical in reproducing experimental properties of flavins including their electronic spectra, vibrational signatures, and nuclear magnetic resonance (NMR) chemical shifts. A computational toolbox for understanding flavin reactivity moreover must be able to treat all five oxidation and protonation states, in addition to excited states that participate in flavoprotein's light-driven reactions. Therefore, we compare emerging hybrid strategies and their successes in replicating effects of hydrogen bonding, the surrounding dielectric, and local electrostatics. These contribute to the protein's ability to modulate flavin reactivity, so we conclude with a survey of methods for incorporating the effects of the protein residues explicitly, as well as local dynamics. Computation is poised to elucidate the factors that affect a bound flavin's ability to mediate stunningly diverse reactions, and make life possible.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat

Spectroscopic and mechanistic investigation of two flavin-dependent enzymes: nitronate monooxygenase and choline oxidase

Propionate 3-nitronate (P3N) is a natural toxin that irreversibly inhibits mitochondrial succinate dehydrogenase. P3N poisoning leads to a variety of neurological disorders and even death. Nitronate monooxygenase (NMO) from Cyberlindnera saturnus (CsNMO) and Pseudomonas aeruginosa PAO1 (PaNMO) serve as paradigms for Class I NMO, which catalyze the oxidation of P3N involving single electron transfer. In this dissertation, the crystallographic structure of CsNMO was solved and demonstrated a highly conserved three-dimensional structure and active site with respect to NMO from PaNMO. The role of conserved residues in the active site of Class I NMO, e.g. Y109, Y254, Y299, Y303, and K307 in PaNMO in substrate binding and catalysis were investigated using site-directed mutagenesis, steady-state kinetics and pH effects on the UV-visible absorption spectrum. The study revealed that a protonated tyrosine is required for binding of the negatively charged P3N substrate. We also report that PaNMO can stabilize both the neutral and anionic semiquinones anaerobically for hours, providing a constant protein environment to study their photochemical and photophysical properties. Choline oxidase catalyzes two-step oxidation of choline to glycine betaine with betaine aldehyde as an intermediate. The FAD cofactor is covalently attached to the choline oxidase via H99 through an 8α-N3-histidyl linkage. In the active site of choline oxidase, S101 and H466 are located on two extent loops, ~ 4 Å from the flavin C4a atom. In this dissertation, a charge-induced, reversible C4a-S-cysteinyl-8α-N3-histidyl FAD was engineered by replacing S101 with a cysteine. The mechanistic rationale for the stabilization of de novo C4a-S-cysteinyl-flavins was illustrated with rapid kinetics, pH, kinetic isotope effects and proton inventory. A photoinduced transient C4a-N-histidyl-8α-N3-histidyl FAD in choline oxidase wild-type was also observed with the aid of fluorescence excitation spectroscopy. Site-directed mutagenesis, solvent equilibrium isotope effects and pH effects on the stoke shifts of flavin in choline oxidase wild-type demonstrated H466 as the adduct on the C4a atom of flavin upon excitation, and provided a mechanistic rationale involving photoinduced electron transfer (PET) for the formation of the novel photoinduced transient flavin C4a adduct

\u3csup\u3e15\u3c/sup\u3eN SOLID-STATE NMR DETECTION OF FLAVIN PERTURBATION BY H-BONDING IN MODELS AND ENZYME ACTIVE SITES

Massey and Hemmerich proposed that the different reactivities displayed by different flavoenzymes could be achieved as a result of dominance of different flavin ring resonance structures in different binding sites. Thus, the FMN cofactor would engage in different reactions when it had different electronic structures. To test this proposal and understand how different protein sites could produce different flavin electronic structures, we are developing solid-state NMR as a means of characterizing the electronic state of the flavin ring, via the 15N chemical shift tensors of the ring N atoms. These provide information on the frontier orbitals. We propose that the 15N chemical shift tensors of flavins engaged in different hydrogen bonds will differ from one another. Tetraphenylacetyl riboflavin (TPARF) is soluble in benzene to over 250 mM, so, this flavin alone and in complexes with binding partners provides a system for studying the effects of formation of specific hydrogen bonds. For N5, the redoxactive N atom, one of the chemical shift principle values (CSPVs) changed 10 ppm upon formation of a hydrogen bonded complex, and the results could be replicated computationally. Thus our DFT-derived frontier orbitals are validated by spectroscopy and can be used to understand reactivity. Indeed, our calculations indicate that the electron density in the diazabutadiene system diminishes upon H-bond complex formation, consistent with the observed 100 mV increase in reduction midpoint potential. Thus, the current studies of TPARF and its complexes provide a useful baseline for further SSNMR studies aimed at understanding flavin reactivity in enzymes

Doctor of Philosophy

dissertationType 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) catalyzes the reversible conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP). The enzyme requires a divalent cation (Mg2+) and a reduced flavin mononucleotide (FMNred) cofactor to be catalytically active. To probe the role of FMNred, substrate analogues were incubated with enzyme-bound FMNred and analyzed under various conditions by UV-vis absorption and fluorescence spectroscopy. The spectral characteristics indicated a covalent bond was formed between the analogues and flavin at either the C4a- or N5-position (dependent on the substrate analogues). Similar methodology was applied to study a flavin adduct that formed when IDI-2 was incubated with IPP or DMAPP. Furthermore, mass spectrometry demonstrated that this adduct was the result of a loss of inorganic diphosphate and yielded a single isoprene unit addition to flavin. Bond formation observed with many analogues and the natural substrates supports a role where flavin is involved with catalysis directly as a proton donor/acceptor and/or as a stabilizer of an intermediate. Additionally, the kinetic mechanism was established for the apo form of Streptococcus pneumoniae IDI-2, a potential antibacterial drug target. Bisubstrate studies (where IPP and FMN were varied) and competitive inhibition experiments (with a competitive inhibitor of both substrates) were employed to determine an ordered sequential mechanism, where FMN binds first. The kinetic data appeared sigmoidal and could only be fit when Hill coefficients where included in the mathematical models, particularly for FMN. The Hill coefficients were thoroughly evaluated statistically and their inclusion was determined to be significant, thus a possible cooperative or allosteric effect occurs upon the binding of FMN. These experiments suggest that allosteric inhibitors could be investigated to inactivate the S. pneumoniae IDI-2 enzyme. In a different application of statistics, transient kinetic isotope effect studies were evaluated using the bootstrap statistical technique. While statistically significant isotope effects were determined, the values were 100 % larger than expected. Thus, the technique was successful and identified an underlying problem with the experimental design during data collection

The Function of the Halophilic Dodecin

Flavins are physiologically relevant cofactors that catalyze various redox and light-induced reactions. Due to a high intrinsic reactivity, these compounds are found tightly bound to proteins with the chemistry of the flavin either narrowed to a defined reaction channel (flavoenzymes) or reduced to (almost) non-reactivity (flavin binding and carrier proteins). Lumichrome is a product of flavin photodegradation. In spite of the structural similarity to flavins, lumichrome has electronic properties which differ from flavins, preventing this compound from any physiological relevance as a cofactor. The interest in lumichrome is basically focussed on its role as a photosensitizing compound. Lumichrome is excited by the absorption of visible light and relaxes by transferring electrons or electronic energy to surrounding substrates and oxygen, exerting an unspecific toxic effect on the cellular environment. Dodecin is a dodecameric flavin binding protein comprising a novel ligand binding fold. It incorporates dimers of ligands arranged in antiparallel manner within each of the six identical binding pockets. In this thesis, structure and function of dodecin from the archaeal organism Halobacterium salinarum are reported. X-ray structural investigations supplemented with functional data revealed that this protein is an unspecific binder of flavins and binder of the flavin-like compound lumichrome. Dissociation constants were obtained in the nanomolar to micromolar range and found to correlate positively with the ligand size. The preference of dodecin for the small ligands lumichrome and lumiflavin is described as a gated ligand binding mode, based on the low plasticity of the dodecin binding pocket which sterically restricts the bulkier ligands from arranging the flavin aromatic subunit in a high affinity position. Site directed mutagenesis of the halophilic dodecin allowed to spread the idea of dodecin as a small ligand binding particle among homologous proteins. These mutational studies could moreover show that the halophilic type of dodecins is outstanding in additionally exhibiting a high affinity for riboflavin. The stabilization of the ribityl chain by an H-bond network to a single residue was found to suspend restrictions of the gated ligand binding mode and to enable H. salinarum dodecin to exhibit multiple (high) affinity. In Western-Blot and RT-PCR analysis of the dodecin expression level, it could be demonstrated that after a short lag period dodecin is constitutively expressed in light and in dark. In the late stationary phase, a clear influence of dodecin on the riboflavin cellular concentrations could be observed. While high levels of riboflavin were found in H. salinarum wild type cells, in cells of the dodecin deficient strain riboflavin cellular concentrations were depressed. Lumichrome concentrations on the other hand were unaffected from dodecin; however; increased concentrations of lumichrome were found in light, according to a photolytic degradation of riboflavin. In vivo data fully agreed with the deductions from the dodecin structural and functional investigations. Dodecin is a riboflavin binding and carrier protein (RfBP). Its function is to store riboflavin under non-favorable environmental conditions while preventing this flavin from photodegradation. The lumichrome-collecting property represents an extra-feature which allows binding of lumichrome if degradation of riboflavin occurs in order to protect the cellular environment from high amounts of this photo-toxic compound

The intrinsic fluorescence of FAD and its application in analytical chemistry: a review

This review (with 106 references) mainly deals with the analytical applications of flavin-adenine dinucleotide (FAD) fluorescence. In the first section, the spectroscopic properties of this compound are reviewed at the light of his different acid-base, oxidation and structural forms; the chemical and spectroscopic properties of flavin mononucleotide (FMN) and other flavins will be also briefly discussed. The second section discusses how the properties of FAD fluorescence changes in flavoenzymes (FvEs), again considering the different chemical and structural forms; the glucose oxidase (GOx) and the choline oxidase (ChOx) cases will be commented. Since almost certainly the most reported analytical application of FAD fluorescence is as an auto-indicator in enzymatic methods catalysed by FvE oxidoreductases, it is important to know how the concentrations of the different forms of FAD changes along the reaction and, consequently, the fluorescence and the analytical signals. An approach to do this will be presented in section 3. The fourth part of the paper compiles the analytical applications which have been reported until now based in these fluorescence properties. Finally, some suggestions about tentative future research are also given