Substrate Dehydrogenation Research Articles

Kinetically restrained oxygen reduction to hydrogen peroxide with nearly 100% selectivity

Hydrogen peroxide has been synthesized mainly through the electrocatalytic and photocatalytic oxygen reduction reaction in recent years. Herein, we synthesize a single-atom rhodium catalyst (Rh1/NC) to mimic the properties of flavoenzymes for the synthesis of hydrogen peroxide under mild conditions. Rh1/NC dehydrogenates various substrates and catalyzes the reduction of oxygen to hydrogen peroxide. The maximum hydrogen peroxide production rate is 0.48 mol gcatalyst−1 h−1 in the phosphorous acid aerobic oxidation reaction. We find that the selectivity of oxygen reduction to hydrogen peroxide can reach 100%. This is because a single catalytic site of Rh1/NC can only catalyze the removal of two electrons per substrate molecule; thus, the subsequent oxygen can only obtain two electrons to reduce to hydrogen peroxide through the typical two-electron pathway. Similarly, due to the restriction of substrate dehydrogenation, the hydrogen peroxide selectivity in commercial Pt/C-catalyzed enzymatic reactions can be found to reach 75%, which is 30 times higher than that in electrocatalytic oxygen reduction reactions.

A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation.

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).

C-H Bond Activation by an Imido Cobalt(III) and the Resulting Amido Cobalt(II) Complex.

The 3d‐metal mediated nitrene transfer is under intense scrutiny due to its potential as an atom economic and ecologically benign way for the directed amination of (un)functionalised C−H bonds. Here we present the isolation and characterisation of a rare, trigonal imido cobalt(III) complex, which bears a rather long cobalt–imido bond. It can cleanly cleave strong C−H bonds with a bond dissociation energy of up to 92 kcal mol−1 in an intermolecular fashion, unprecedented for imido cobalt complexes. This resulted in the amido cobalt(II) complex [Co(hmds)2(NHtBu)]−. Kinetic studies on this reaction revealed an H atom transfer mechanism. Remarkably, the cobalt(II) amide itself is capable of mediating H atom abstraction or stepwise proton/electron transfer depending on the substrate. A cobalt‐mediated catalytic application for substrate dehydrogenation using an organo azide is presented.

Replacement of Stoichiometric DDQ with a Low Potential o-Quinone Catalyst Enabling Aerobic Dehydrogenation of Tertiary Indolines in Pharmaceutical Intermediates.

A transition-metal/quinone complex, [Ru(phd)3]2+ (phd = 1,10-phenanthroline-5,6-dione), is shown to be effective for aerobic dehydrogenation of 3° indolines to the corresponding indoles. The results show how low potential quinones may be tailored to provide a catalytic alternative to stoichiometric DDQ, due to their ability to mediate efficient substrate dehydrogenation while also being compatible with facile reoxidation by O2. The utility of the method is demonstrated in the synthesis of key intermediates to pharmaceutically important molecules.

C–H Oxidation by H2O2 and O2 Catalyzed by a Non-Heme Iron Complex with a Sterically Encumbered Tetradentate N-Donor Ligand

The compound N,N'-dineopentyl-N,N'-bis(2-pyridylmethyl)-1,2-ethanediamine (dnbpn) and its ferrous complex [Fe(dnbpn)(OTf)2] were synthesized. The Fe(II) complex was used to catalyze the oxidation of hydrocarbons by H2O2 and O2. Although the catalyzed alkane oxidation by H2O2 displays a higher preference for secondary over tertiary carbons than those associated with most previously reported nonheme iron catalysts, the catalytic activity is markedly inferior. In addition to directing the catalyzed oxidation toward the less sterically congested C-H bonds of the substrates, the neopentyl groups destabilize the metal-based oxidants generated from H2O2 and the Fe(II) complex. The presence of benzylic substrates with weak C-H bonds stabilizes an intermediate which we have tentatively assigned as a high-spin ferric hydroperoxide species. The oxidant generated from O2 reacts with allylic and benzylic C-H bonds in the absence of a sacrificial reductant; less substrate dehydrogenation is observed than with related previously described systems that use O2 as a terminal oxidant.

The Mechanism of Borane–Amine Dehydrocoupling with Bifunctional Ruthenium Catalysts

Borane-amine adducts have received considerable attention, both as vectors for chemical hydrogen storage and as precursors for the synthesis of inorganic materials. Transition metal-catalyzed ammonia-borane (H3N-BH3, AB) dehydrocoupling offers, in principle, the possibility of large gravimetric hydrogen release at high rates and the formation of B-N polymers with well-defined microstructure. Several different homogeneous catalysts were reported in the literature. The current mechanistic picture implies that the release of aminoborane (e.g., Ni carbenes and Shvo's catalyst) results in formation of borazine and 2 equiv of H2, while 1 equiv of H2 and polyaminoborane are obtained with catalysts that also couple the dehydroproducts (e.g., Ir and Rh diphosphine and pincer catalysts). However, in comparison with the rapidly growing number of catalysts, the amount of experimental studies that deal with mechanistic details is still limited. Here, we present a comprehensive experimental and theoretical study about the mechanism of AB dehydrocoupling to polyaminoborane with ruthenium amine/amido catalysts, which exhibit particularly high activity. On the basis of kinetics, trapping experiments, polymer characterization by (11)B MQMAS solid-state NMR, spectroscopic experiments with model substrates, and density functional theory (DFT) calculations, we propose for the amine catalyst [Ru(H)2PMe3{HN(CH2CH2PtBu2)2}] two mechanistically connected catalytic cycles that account for both metal-mediated substrate dehydrogenation to aminoborane and catalyzed polymer enchainment by formal aminoborane insertion into a H-NH2BH3 bond. Kinetic results and polymer characterization also indicate that amido catalyst [Ru(H)PMe3{N(CH2CH2PtBu2)2}] does not undergo the same mechanism as was previously proposed in a theoretical study.

Cholesterol oxidase: biochemistry and structural features

Cholesterol oxidases are bifunctional flavoenzymes that catalyze the oxidation of steroid substrates which have a hydroxyl group at the 3beta position of the steroid ring system. The enzyme is found, in a wide range of bacterial species, in two forms: one with the FAD cofactor bound noncovalently to the enzyme; and one with the cofactor linked covalently to the protein. Here we discuss, compare and contrast the salient biochemical properties of the two forms of the enzyme. Specifically, the structural features are discussed that affect the redox potentials of the flavin cofactor, the chemical mechanism of substrate dehydrogenation by active-center amino acid residues, the kinetic parameters of both types of enzymes and the reactivity of reduced enzymes with molecular dioxygen. The presence of a molecular tunnel that is proposed to serve in the access of dioxygen to the active site and mechanisms of its control by a 'gate' formed by amino acid residues are highlighted.

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production.

Oxygen consumption and metabolite concentrations during transitions between different work intensities in heart

Steady-state metabolite (ADP, ATP, P(i), PCr, and NADH) concentrations usually differ little between different workloads with significantly different oxygen consumption rates in the heart. However, during transitions between steady states, metabolite concentrations may in some cases change transiently, exhibiting a significant overshoot or undershoot, whereas in other cases they approach near-exponentially new steady-state values. Oxygen consumption rate usually reaches the new steady-state value very quickly (within a few seconds). The present in silico studies, performed using a previously developed computer model of oxidative phosphorylation in the heart, demonstrate that such a behavior of the oxidative phosphorylation system can be reproduced only under the assumption that ATP usage, substrate dehydrogenation, and (particular steps of) oxidative phosphorylation are directly activated to a similar extend by some cytosolic factor/mechanism during transition from low work to high work (the so-called parallel-activation mechanism). Computer simulations show that some differences observed between different experimental systems can be explained by a slightly different balance of the activation of particular components of the system and/or by a delay in time of the activation/inactivation of substrate dehydrogenation and oxidative phosphorylation during low-to-high and high-to-low work transitions. Thus the presented theoretical approach offers a general idea that is able to unify, at least semiquantitatively, different experimental data available in the literature.

Calcium-mediated coupling between mitochondrial substrate dehydrogenation and cardiac workload in single guinea-pig ventricular myocytes

We measured mitochondrial NADH autofluorescence or Ca(2+) using Rhod-2, simultaneously with cell shortening in isolated guinea-pig ventricular myocytes. When both frequency and amplitude of twitch shortening (work intensity) were increased by raising stimulus frequency in incremental steps from 0.1 to 3.3 Hz, the steady level of NADH signal increased in a frequency-dependent manner. Mitochondrial Ca(2+) also increased with increasing work intensity. Applying Ru360, an inhibitor of mitochondrial Ca(2+) uniporter, largely attenuated the response of both NADH fluorescence and mitochondrial Ca(2+). The increase in mitochondrial Ca(2+) was slow with t(1/2)=~12 s and no obvious cyclic changes were observed in the NADH signal. When a step change from 0.1 to 3.3 Hz stimulation was applied, the NADH signal first decreased to 83% and then increased to 155% of the control level. Upon returning to 0.1 Hz, the NADH signal showed an overshoot before declining to the control level. The biphasic onset time course was well explained by the delayed Ca(2+) activation of the substrate dehydrogenation superimposed on the feedback control of the ATP synthesis, while the offset time course with a delayed deactivation of dehydrogenation. A computer simulation using an oxidative phosphorylation linked to the cardiac excitation contraction model well reconstructed the response of NADH. This model simulation predicts that the activation of substrate dehydrogenation provides ~23% of driving force of the ATP synthesis to meet the increased workload induced by the jump of stimulus from 0.1 to 3.3 Hz, and remaining ~77% is supplied by the feedback control.

Regulation of oxidative phosphorylation in intact mammalian heart in vivo

A dynamic computer model of oxidative phosphorylation in intact heart was developed by modifying the model of oxidative phosphorylation in intact skeletal muscle published previously. Next, this model was used for theoretical studies on the regulation of oxidative phosphorylation in intact heart in vivo during transition between different work intensities. It is shown that neither a direct activation of ATP usage alone nor a direct activation of both ATP usage and substrate dehydrogenation, including the calcium-activated tricarboxylate acid cycle dehydrogenases, can account for the constancy of [ADP], [PCr], [P i] and [NADH] during a significant increase in oxygen consumption and ATP turnover encountered in intact heart in vivo. Only a direct activation of all oxidative phosphorylation complexes in parallel with a stimulation of ATP usage and substrate dehydrogenation enabled to reproduce the experimental data concerning the constancy of metabolite concentrations. The molecular background of the differences between heart and skeletal muscle in the kinetic behaviour of the oxidative phosphorylation system is also discussed.

The Modeling of Oxidative Phosphorylation in Skeletal Muscle

A computer model of oxidative phosphorylation was developed in isolated muscle mitochondria [Korzeniewski and Mazat: Biochem J 319: 143-148, 1996] and in intact skeletal muscle [Korzeniewski and Zoladz: Biophys Chem 92: 17-34, 2001]. Within this model the dependence on different metabolite concentrations of the rate of each enzymatic reaction, process and flux is described by an appropriate kinetic equation. The changes of metabolite concentrations over time are described by a set of ordinary differential equations. The model has been very extensively tested by a comparison of computer simulations with a broad set of experimental results concerning various kinetic properties of the oxidative phosphorylation system. Next the model was used for theoretical studies on the regulation of oxidative phosphorylation in intact muscle cells. The model decidedly supports the so-called parallel-activation mechanism or each-step-activation mechanism of adjusting the rate of ATP supply to the current energy demand [Korzeniewski: Biochem J 330: 1189-1195, 1998; Korzeniewski: Biochem J 375: 799-804, 2003]. Because of this mechanism, not only ATP usage, but also the substrate dehydrogenation system and all oxidative phosphorylation complexes (complex I, complex III, complex IV, ATP synthase, ATP/ADP carrier, phosphate carrier) are directly (and not by changes in metabolite concentrations) activated by some intracellular factor(s) related to muscle contraction, probably by calcium ions, during the transition from rest to work. This mechanism is able to account for several kinetic properties of oxidative phosphorylation that cannot be explained by other mechanisms postulated in the literature. Thus the discussed kinetic model of oxidative phosphorylation has appeared to be a very useful research tool.

The catalytic mechanism of Drosophila alcohol dehydrogenase: evidence for a proton relay modulated by the coupled ionization of the active site Lysine/Tyrosine pair and a NAD+ ribose OH switch.

The ionization properties of the active site residues in Drosophila lebanonensis alcohol dehydrogenase (DADH) were investigated theoretically by using an approach developed to account for multiple locations of the hydrogen atoms of the titratable and polar groups. The electrostatic calculations show that (a) the protonation/deprotonation transition of the binary complex of DADH is related to the coupled ionization of Tyr151 and Lys155 in the active site and (b) the pH dependence of the proton abstraction is correlated with a reorganization of the hydrogen bond network in the active site. On this basis, a proton relay mechanism for substrate dehydrogenation is proposed in which the O2' ribose hydroxyl group from the coenzyme has a key role and acts as a switch. The proton relay chain includes the active site catalytic residues, as well as a chain of eight water molecules that connects the active site with the bulk solvent.

PH and kinetic isotope effects in d-amino acid oxidase catalysis.

The effects of pH, solvent isotope, and primary isotope replacement on substrate dehydrogenation by Rhodotorula gracilis d-amino acid oxidase were investigated. The rate constant for enzyme-FAD reduction by d-alanine increases approximately fourfold with pH, reflecting apparent pKa values of approximately 6 and approximately 8, and reaches plateaus at high and low pH. Such profiles are observed in all presteady-state and steady-state kinetic experiments, using both d-alanine and d-asparagine as substrates, and are inconsistent with the operation of a base essential to catalysis. A solvent deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with d-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary substrate isotope effect on the reduction rate with [2-D]d-alanine is 9.1 +/- 1.5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with [2-D]d-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) are independent of the presence of the other isotope, i.e. the 'double' kinetic isotope effect is the product of the individual KIEs, consistent with a transition state in which rupture of the two bonds of the substrate to hydrogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in d-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pKa,app of approximately 8 is interpreted to arise from the microscopic ionization of the substrate amino acid alpha-amino group, but also includes contributions from kinetic parameters.

ChemInform Abstract: Substrate Dehydrogenation by Flavoproteins

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Substrate dehydrogenation by flavoproteins.

Enzymes with tightly bound FMN or FAD as cofactor catalyze the oxidation of a wide range of substrates. The chemical versatility of the isoalloxazine ring provides these enzymes with a range of potential mechanisms. Recent progress in elucidating the mechanisms of oxidation of organic substrates by flavoenzymes is described, focusing on the oxidation of alcohols, amino and hydroxy acids, amines, and nitroalkanes. With each family of enzymes, an attempt is made to integrate mechanistic, structural, and biomimetic data into a common catalytic mechanism.

The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.

Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

Mechanism of activation of acyl-CoA substrates by medium chain acyl-CoA dehydrogenase: interaction of the thioester carbonyl with the flavin adenine dinucleotide ribityl side chain.

The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acyl-coenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3'-deoxy-FAD and ribityl-2'-deoxy-FAD. 3'-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3'-OH group does not play any particular role in cofactor binding or catalysis. 2'-Deoxy-FAD-MCADH was characterized using the natural substrate C8CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2'-deoxy-FAD-MCADH is approximately 1.5 x 10(7)-fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2'-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to approximately 38 kJ mol-1. The alphaC-H microscopic pKa of the substrate analogue 3S-C8CoA, which undergoes alpha-deprotonation on binding to MCADH, is lowered from approximately 16 in the free state to approximately 11 (+/-0.5) when bound to 2'-deoxy-FAD-MCADH. This compares with a decrease of the same pKa to approximately 5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of approximately 11 pK units, i.e., approximately 65 kJ mol-1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of approximately 6 pK units ( approximately 35 kJ mol-1) between MCADH and 2'-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2'-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of alphaC-H microscopic pKa of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2'-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2'-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2'-OH hydrogen bond. The large energy associated with the 2'-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.

Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial deficiencies.

1. The dynamic model of oxidative phosphorylation developed previously for rat liver mitochondria incubated with succinate was adapted for muscle mitochondria respiring on pyruvate. We introduced the following changes considering: (1) a higher external ATP/ADP ratio and an ATP/ADP carrier less displaced from equilibrium; (2) a substrate dehydrogenation more sensitive to the NADH/NAD+ ratio; and (3) the respiratory chain, ATP synthase and phosphate carrier being more displaced from equilibrium. The experimental flux control coefficients already determined in state 3 for respiratory rate and ATP synthesis were used to adjust some parameters. This new oxidative phosphorylation model enabled us to simulate the whole titration curves obtained experimentally in state 3. These curves, which mimic the effect of mitochondrial complex deficiencies on oxidative phosphorylation, show a threshold effect, which is reproduced by the model. 2. the model was also used to simulate other physiological conditions such as (i) state 3.5, conditions in-between state 4 and state 3; and (ii) hypoxic conditions. In both cases a profound change in the pattern of the control coefficients was shown. 3. This model was thus found useful in investigating a variety of new conditions, the most interesting of which can then be experimentally studied.

Simulation of oxidative phosphorylation in hepatocytes

The dynamic mathematical model of oxidative phosphorylation proposed previously was modified, developed and further tested. The description of cytochrome oxidase kinetics was changed to involve dependence on Δp. Simple, phenomenological descriptions of the kinetics of substrate dehydrogenation and ATP usage, able to reflect experimental data correctly, were found. The kinetic response of the oxidation subsystem (substrate dehydrogenation, respiratory chain), phosphorylation subsystem (ATP synthase, ATP ADP carrier, phosphate carrier, ATP usage) and proton leak to the changes of Δp in isolated hepatocytes incubated with different respiratory substrates was simulated. The simulations revealed a good agreement with the experimental results. Simple, intuitive assumptions were able, when introduced into the model, to explain differences in the properties of the oxidative phosphorylation system working with different respiratory substrates. It was proposed, therefore, that our explicit understanding of the oxidative phosphorylation system was good enough to explain many properties of this system correctly, at least in the range of physiological conditions tested.