Insights on the mechanism of amine oxidation catalyzed by D-arginine dehydrogenase through pH and kinetic isotope effects

The mechanism of amine oxidation catalyzed by D-arginine dehydrogenase (DADH) has been investigated using steady-state and rapid reaction kinetics, with pH, substrate and solvent deuterium kinetic isotope effects (KIE) as mechanistic probes, and computational studies. Previous results showed that 85-90% of the flavin reduction reaction occurs in the mixing time of the stopped-flow spectrophotometer when arginine is the substrate, precluding a mechanistic investigation. Consequently, leucine, with slower kinetics, has been used here as the flavin-reducing substrate. Free energy calculations and the pH profile of the K(d) are consistent with the enzyme preferentially binding the zwitterionic form of the substrate. Isomerization of the Michaelis complex, yielding an enzyme-substrate complex competent for flavin reduction, is established due to an inverse hyperbolic dependence of k(cat)/K(m) on solvent viscosity. Amine deprotonation triggers the oxidation reaction, with cleavage of the substrate NH and CH bonds occurring in an asynchronous fashion, as suggested by the multiple deuterium KIE on the rate constant for flavin reduction (k(red)). A pK(a) of 9.6 signifies the ionization of a group that facilitates flavin reduction in the unprotonated form. The previously reported high-resolution crystal structures of the iminoarginine and iminohistidine complexes of DADH allow us to propose that Tyr(53), on a mobile loop covering the active site, may participate in substrate binding and facilitate flavin reduction.     

PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF Euglena grcrcilis

Abstract —Phototaxis of the flagellate Euglena gracilis has been thought to be mediated by flavin photoreceptor molecules localized in the paraflagellar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-binding protein was solubilized and purified using nonionic detergents, high ionic strength, affinity Chromatography and standard column separations. Sodium dodecyl sulfate gel electrophoresis showed an apparent molecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K_D= 4 n M ) as well as under reducing conditions in the presence of dithionite (K_d= 6 n M ). Affinities towards flavin mononucleotide and flavin adenine dinucleotide were lower. Based on binding data and on estimates of the purified 68 kDa polypeptide, approximately lo⁶ flavin-binding sites were determined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.     

Photoexcitation of the blue light using FAD photoreceptor AppA results in ultrafast changes to the protein matrix

Photoexcitation of the flavin chromophore in the BLUF photosensor AppA results in a conformational change that leads to photosensor activation. This conformational change is mediated by a hydrogen-bonding network that surrounds the flavin, and photoexcitation is known to result in changes in the network that include a strengthening of hydrogen bonding to the flavin C4═O carbonyl group. Q63 is a key residue in the hydrogen-bonding network, and replacement of this residue with a glutamate results in a photoinactive mutant. While the ultrafast time-resolved infrared (TRIR) spectrum of Q63E AppA(BLUF) is characterized by flavin carbonyl modes at 1680 and 1650 cm(-1), which are similar in frequency to the analogous modes from the light activated state of the wild-type protein, a band is also observed in the TRIR spectrum at 1724 cm(-1) that is unambiguously assigned to the Q63E carboxylic acid based on U-(13)C labeling of the protein. Light absorption instantaneously (<100 fs) bleaches the 1724 cm(-1) band leading to a transient absorption at 1707 cm(-1). Because Q63E is not part of the isoalloxazine electronic transition, the shift in frequency must arise from a sub picosecond perturbation to the flavin binding pocket. The light-induced change in the frequency of the Q63E side chain is assigned to an increase in hydrogen-bond strength of 3 kcal mol(-1) caused by electronic reorganization of the isoalloxazine ring in the excited state, providing direct evidence that the protein matrix of AppA responds instantaneously to changes in the electronic structure of the chromophore and supporting a model for photoactivation of the wild-type protein that involves initial tautomerization of the Q63 side chain.     

Characterization of the two-component, FAD-dependent monooxygenase SgcC that requires carrier protein-tethered substrates for the biosynthesis of the enediyne antitumor antibiotic C-1027

C-1027 is a potent antitumor antibiotic composed of an apoprotein (CagA) and a reactive enediyne chromophore. The chromophore has four distinct chemical moieties, including an ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety, the biosynthesis of which from l-alpha-tyrosine requires five proteins: SgcC, SgcC1, SgcC2, SgcC3, and SgcC4; a sixth protein, SgcC5, catalyzes the incorporation of this beta-amino acid moiety into C-1027. Biochemical characterization of SgcC has now revealed that (i) SgcC is a two-component, flavin adenine dinucleotide (FAD)-dependent monooxygenase, (ii) SgcC is only active with SgcC2 (peptidyl carrier protein)-tethered substrates, (iii) SgcC-catalyzed hydroxylation requires O 2 and FADH 2, the latter supplied by the C-1027 pathway-specific flavin reductase SgcE6 or Escherichia coli flavin reductase Fre, and (iv) SgcC efficiently catalyzes regioselective hydroxylation of 3-substituted beta-tyrosyl-S-SgcC2 analogues, including the chloro-, bromo-, iodo-, fluoro-, and methyl-substituted analogues, but does not accept 3-hydroxy-beta-tyrosyl-S-SgcC2 as a substrate. Together with the in vitro data for SgcC4, SgcC1, and SgcC3, the results establish that SgcC catalyzes the hydroxylation of ( S)-3-chloro-beta-tyrosyl-S-SgcC2 as the final step in the biosynthesis of the ( S)-3-chloro-5-hydroxy-beta-tyrosine moiety prior to incorporation into C-1027. SgcC now represents the first biochemically characterized two-component, FAD-dependent monooxygenase that acts on a carrier-protein-tethered aromatic substrate.     

Covalent Attachment of Active Enzymes to Upconversion Phosphors Allows Ratiometric Detection of Substrates

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface-accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide-thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state.     

Primary events in the blue light sensor plant cryptochrome: intraprotein electron and proton transfer revealed by femtosecond spectroscopy

Photoreceptors are chromoproteins that undergo fast conversion from dark to signaling states upon light absorption by the chromophore. The signaling state starts signal transduction in vivo and elicits a biological response. Therefore, photoreceptors are ideally suited for analysis of protein activation by time-resolved spectroscopy. We focus on plant cryptochromes which are blue light sensors regulating the development and daily rhythm of plants. The signaling state of these flavoproteins is the neutral radical of the flavin chromophore. It forms on the microsecond time scale after light absorption by the oxidized state. We apply here femtosecond broad-band transient absorption to early stages of signaling-state formation in a plant cryptochrome from the green alga Chlamydomonas reinhardtii. Transient spectra show (i) subpicosecond decay of flavin-stimulated emission and (ii) further decay of signal until 100 ps delay with nearly constant spectral shape. The first decay (i) monitors electron transfer from a nearby tryptophan to the flavin and occurs with a time constant of τ(ET) = 0.4 ps. The second decay (ii) is analyzed by spectral decomposition and occurs with a characteristic time constant τ(1) = 31 ps. We reason that hole transport through a tryptophan triad to the protein surface and partial deprotonation of tryptophan cation radical hide behind τ(1). These processes are probably governed by vibrational cooling. Spectral decomposition is used together with anisotropy to obtain the relative orientation of flavin and the final electron donor. This narrows the number of possible electron donors down to two tryptophans. Structural analysis suggests that a set of histidines surrounding the terminal tryptophan may act as proton acceptor and thereby stabilize the radical pair on a 100 ps time scale.     

Model systems for flavoenzyme activity: site-isolated redox behavior in flavin-functionalized random polystyrene copolymers

[reaction: see text] A model system has been developed to study the redox behaviors of flavin derivatives appended onto random polystyrene copolymers through "click" chemistry strategies. The results demonstrate that flavin units attached onto polymers exhibit site-isolated redox behaviors, yielding new materials with electrochemically tunable associations (K(a)(ox) = 450 M(-)(1), K(a)(red) = 18,200 M(-)(1)) to complementary diamidopyridine (DAP) functionality.     

One- and Two-Electron Reductions in MiniSOG and their Implication in Catalysis**

The unconventional bioorthogonal catalytic activation of anticancer metal complexes by flavin and flavoproteins photocatalysis has been reported recently. The reactivity is based on a two-electron redox reaction of the photoactivated flavin. Furthermore, when it comes to flavoproteins, we recently reported that site mutagenesis can modulate and improve this catalytic activity in the mini Singlet Oxygen Generator protein (SOG). In this paper, we analyze the reductive half-reaction in different miniSOG environments by means of density functional theory. We report that the redox properties of flavin and the resulting reactivity of miniSOG is modulated by specific mutations, which is in line with the experimental results in the literature. This modulation can be attributed to the fundamental physicochemical properties of the system, specifically (i) the competition of single and double reduction of the flavin and (ii) the probability of electron transfer from the protein to the flavin. These factors are ultimately linked to the stability of flavin‘s electron-accepting orbitals in different coordination modes.     

Towards zirconium phosphonate-based microarrays for probing DNA-protein interactions: critical influence of the location of the probe anchoring groups

Terminal phosphate groups on double-stranded DNA probes bind strongly to glass substrates coated with a zirconium phosphonate monolayer, and probes immobilized in this way as microarrays can be used to detect protein targets. The sensitivity of the microarray was shown to be enhanced by the use of a polyguanine segment ((G)n , n > or = 5) as a spacer between the phosphate linker and the protein interaction domain. More importantly, the presence of phosphate linkers on both ends of the dsDNA probes leads to significant enhancement of target capture. The relevant characteristics of the different probes when bound to the surface were determined, by the original use of a combination of surface characterization techniques (XPS, AFM, and Sarfus). In this context, the location of the phosphate linkers in the duplex probes was found to result in different probe surface coverage and presentation on the surface, which affect subsequent interactions with the target protein.     

A FLAVOPROTEIN MAY MEDIATE THE BLUE LIGHT-ACTIVATED BINDING OF GUANOSINE 5'-TRIPHOSPHATE TO ISOLATED PLASMA MEMBRANES OF Pisum sativum L.*

Abstract— A blue light photoreceptor has not been identified in higher plants. Most proposals for a blue light-absorbing chromophore lack evidence for a direct connection between the putative chromophdre and a biological effect. Fluorescence data for the plasma membrane from etiolated buds of Pisum sativum L. suggest that we are measuring fluorescence emission of flavin species, and probably not pterin species. Fluorescence data indicate that a putative flavin exists associated with a protein or protein complex in the plasma membrane. Excitation of plasma membranes that were boiled in the presence of 0.1% sodium dodecyl sulfate and treated with blue light yields a fluorescence band with a maximum of approximately 552 nm. This fluorescence emission can be rapidly quenched by the flavin antagonists phenylacetic acid (PAA) and KI. Blue light-enhanced binding of guanosine 5'-[Γ-thio]triphosphate (GTPγS) to a protein in the plasma membrane is strongly inhibited by PAA, KI, and NaN₃, all quenchers of flavin excited states, indicating that a chromophore for this photoreaction may be a flavin associated with a plasma membrane protein. The above evidence is consistent with the participation of a flavin as the chromophore for the light-induced GTP-binding reaction in pea plasma membrane.     

A laser photolysis study of the decay kinetics of the triplet states and radicals of flavins in the bovine eye lens

The decay kinetics of the triplet states and radicals of riboflavin and flavin mononucleotide, which were introduced into the bovine eye lens as kinetic photochemical probes, was studied by nanosecond laser photolysis, as a function of dilution of the lens with water. Correlations were found between the kinetic parameters and the degree of ordering of crystallins in the aqueous solution of the lens.     

Metal substrate catalysis in the confined space for platinum drug delivery

Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min⁻¹) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin.

Loading of a flavin catalyst and Pt prodrug onto a hydrogel affords biomaterials for the catalytic generation and delivery of cisplatin upon light irradiation or addition of electron donors. Confinement boosts the turnover frequency of the flavin.     

Ab initio quantum chemical investigation of the first steps of the photocycle of phototropin: a model study

Phototropin is a blue light-activated photoreceptor that plays a dominant role in the phototropism of plants. The protein contains two subunits that bind flavin mononucleotide (FMN), which are responsible for the initial steps of the light-induced reaction. It has been proposed that the photoexcited flavin molecule adds a cysteine residue of the protein backbone, thus activating autophosphorylation of the enzyme. In this study, the electronic properties of several FMN-related compounds in different charge and spin states are characterized by means of ab initio quantum mechanical calculations. The model compounds serve as idealized model chromophores for phototropism. Reaction energies are estimated for simple model reactions, roughly representing the addition of a cysteine residue to the flavin molecule. Excitation energies were calculated with the help of time-dependent density functional theory. On the basis of these calculations we propose the following mechanism for the addition reaction: (1) after photoexcitation of FMN out of the singlet ground state S0, excited singlet state(s) are populated; these relax to the lowest excited singlet state S1, and subsequently by intersystem crossing FMN in the lowest triplet state, T1 is formed; (2) the triplet easily removes the neutral hydrogen atom from the H-S group of the cysteine residue; and (3) the resulting thio radical is added.     

Evaluation of dsDNA as a wire for redox-active proteins

The redox-active multiligand-binding flavoprotein dodecin binds flavins with high affinity when they are oxidized, whereas flavin reduction induces the dissociation of the holoprotein complex in apododecin and free flavin ligands. Dodecin could be reconstituted at flavin-terminated dsDNA monolayers. The binding and release of apododecin triggered by the redox state of the flavins can be monitored by surface-sensitive techniques such as surface plasmon resonance and quartz crystal microbalance measurements with dissipation monitoring. It has been shown that flavin reduction followed by the release of apododecin can be achieved by mediated electron transfer in the presence of the redox mediator amino ethyl viologen and by chemical flavin reduction, whereas flavin reduction by direct electron transfer via the dsDNA tethers is not possible. The combination of electrochemistry with surface-sensitive techniques such as surface plasmon resonance or quartz crystal microbalance measurements with dissipation monitoring could be highly beneficial to confirm or disprove the mechanism, which has been postulated for the action of primases, which contain a [4Fe4S] cluster and are involved in DNA replication. It has been postulated that these enzymes bind the DNA template when the cluster is in the [4Fe4S]³⁺ state, whereas they are released when the cluster is reduced via electron transfer through DNA and the protein environment.     

LOV-like flavin-Cys adduct formation by introducing a Cys residue in the BLUF domain of TePixD

BLUF and LOV are blue-light sensor domains that possess flavin as a common chromophore but exhibit distinct photoreactions. Ile66 located in the BLUF domain of a cyanobacterial photosensor protein, TePixD, was replaced with Cys to mimic the LOV domain. Light-induced Fourier transform infrared spectra of the I66C TePixD showed that a flavin-Cys adduct, typical of the photoinduced intermediates of LOV domains, was formed in the I66C BLUF domain. This result demonstrates that different types of flavin photoreactions can be realized in the same domain if key amino acids are properly arranged near the flavin and the domain structure itself is not a crucial factor to determine the photoreaction type.     

Near-infrared fluorescent probes: a next-generation tool for protein-labeling applications

The development of near-infrared (NIR) fluorescent probes over the past few decades has changed the way that biomolecules are imaged, and thus represents one of the most rapidly progressing areas of research. Presently, NIR fluorescent probes are routinely used to visualize and understand intracellular activities. The ability to penetrate tissues deeply, reduced photodamage to living organisms, and a high signal-to-noise ratio characterize NIR fluorescent probes as efficient next-generation tools for elucidating various biological events. The coupling of self-labeling protein tags with synthetic fluorescent probes is one of the most promising research areas in chemical biology. Indeed, at present, protein-labeling techniques are not only used to monitor the dynamics and localization of proteins but also play a more diverse role in imaging applications. For instance, one of the dominant technologies employed in the visualization of protein activity and regulation is based on protein tags and their associated NIR fluorescent probes. In this mini-review, we will discuss the development of several NIR fluorescent probes used for various protein-tag systems.

This minireview describes the development of NIR chemical probes for various protein-tag systems.     

Type II isopentenyl diphosphate isomerase: irreversible inactivation by covalent modification of flavin

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic building blocks of isoprenoid molecules. Two structurally unrelated classes of IDI are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction; whereas, the type II enzyme (IDI-2) requires reduced flavin. Epoxy, diene, and fluorinated substrate analogues, irreversible inhibitors of IDI-1, were analyzed as mechanistic probes for IDI-2. 3,4-Oxido-3-methyl-1-butyl diphosphate (eIPP), 3-methylene-4-penten-1-yl diphosphate (vIPP), and 3-(fluoromethyl)-3-buten-1-yl diphosphate (fmIPP) inactivate IDI-2 through formation of covalent adducts with the reduced flavin. UV-visible spectra of the inactivated complexes are consistent with modification of the isoalloxazine ring at position N5. vIPP and fmIPP are also alternate substrates with isomerization competing with alkylation of the flavin cofactor. (Z)-3-(Fluoromethyl)-2-buten-1-yl diphosphate ((Z)-fmDMAPP) and (Z)-3-(difluoromethyl)-2-buten-1-yl diphosphate ((Z)-dfmDMAPP) are alternate substrates, which are isomerized to the corresponding IPP derivatives. The rates of isomerization of fmIPP and (Z)-fmDMAPP are approximately 50-fold less than IPP and DMAPP, respectively. dfmIPP is not an irreversible inhibitor. These studies indicate that the irreversible inhibitors inactivate the reduced flavin required for catalysis by electrophilic alkylation and are consistent with a protonation-deprotonation mechanism for the isomerization catalyzed by IDI-2.     

Modifying the collagen framework of costal cartilage under the impact of UV and a flavin mononucleotide

Modifications of the matrix of the tissue of costal cartilage under the impact of UV (λ = 365 nm) and a flavin mononucleotide (FMN) is studied. The changes in the macroscopic properties of the tissue are detected by means of differential scanning calorimetry and under the conditions of uniaxial compression during mechanical testing. The endothermic effects of the denaturation of the collagen framework of the tissue and the Young’s modulus are determined. It is shown that the presence of a flavin mononucleotide in the interstitial fluid leads lowers the temperature of collagen denaturation by 2.5°С and doubles the Young’s modulus. It is found that the temperature of denaturation and the Young’s modulus grow gradually after treating the tissue with the UV radiation, and their values ultimately exceed by far the corresponding values for intact samples. It is concluded that the obtained data indicate the possibility of stabilizing the framework of the matrix of costal cartilage under the impact of UV radiation and a flavin mononucleotide.     

Recent advances in activity-based probes (ABPs) and affinity-based probes (AfBPs) for profiling of enzymes

Activity-based protein profiling (ABPP) is a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins. ABPP integrates the strengths of both chemical and biological disciplines. By utilizing chemically synthesized or modified bioactive molecules, ABPP is able to reveal complex physiological and pathological enzyme–substrate interactions at molecular and cellular levels. It is also able to provide critical information of the catalytic activity changes of enzymes, annotate new functions of enzymes, discover new substrates of enzymes, and allow real-time monitoring of the cellular location of enzymes. Based on the mechanism of probe-enzyme interaction, two types of probes that have been used in ABPP are activity-based probes (ABPs) and affinity-based probes (AfBPs). This review highlights the recent advances in the use of ABPs and AfBPs, and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.

This review highlights the recent advances in the use of activity-based probes (ABPs) and affinity-based probes (AfBPs), and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.