Biomedical subjects

Jaswir Basran

Publications and source records attributed to Jaswir Basran.

10 recordsLinked to original sources

PubMed2004

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex.

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

Acyl-CoA Dehydrogenase↗

PubMed2003

H-tunneling in the multiple H-transfers of the catalytic cycle of morphinone reductase and in the reductive half-reaction of the homologous pentaerythritol tetranitrate reductase.

The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.

Bacterial Proteins↗

PubMed2003

Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase.

Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes dimethylglycine to a labile iminium intermediate. The C-terminal region binds tetrahydrofolate, comprises three domains arranged in a ring-like structure and is related to the T-protein of the glycine cleavage system. The complex with folinic acid indicates that this enzyme selectively activates the N10 amino group for initial attack on the substrate. Dead-end reactions with oxidized folate are avoided by the strict stereochemical constraints imposed by the folate-binding funnel. The active sites in DMGO are approximately 40 A apart, connected by a large irregular internal cavity. The tetrahydrofolate-binding funnel serves as a transient entry-exit port, and access to the internal cavity is controlled kinetically by tetrahydrofolate binding. The internal cavity enables sequestration of the reactive iminium intermediate prior to reaction with tetrahydrofolate and avoids formation of toxic formaldehyde. This mode of channelling in DMGO is distinct from other channelling mechanisms.

Amino Acid Sequence↗

PubMed2003

Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase.

The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction. Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. Cyanide does not activate turnover in M. methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme. Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations. PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding. Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites. Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration. The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites. Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover. At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting. A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data. The limiting rate of PQQ reduction (0.48 s(-1)) is less than the steady-state turnover number, and the observed KIE in stopped-flow studies is unity. Although catalytically active, we propose reduction of the oxidized enzyme generated in stopped-flow analyses is gated by conformational change or ligand exchange. Slow recovery from this trapped state on mixing with methanol accounts for the slow reduction of PQQ and a KIE of 1. This study emphasizes the need for caution in using inflated KIEs, and the temperature dependence of KIEs, as a probe for hydrogen tunneling.

Alcohol Oxidoreductases↗

PubMed2003

Molecular dissection of human methionine synthase reductase: determination of the flavin redox potentials in full-length enzyme and isolated flavin-binding domains.

Human methionine synthase reductase (MSR) catalyzes the NADPH-dependent reductive methylation of methionine synthase. MSR is 78 kDa flavoprotein belonging to a family of diflavin reductases, with cytochrome P450 reductase (CPR) as the prototype. MSR and its individual flavin-binding domains were cloned as GST-tagged fusion proteins for expression and purification from Escherichia coli. The isolated flavin domains of MSR retain UV-visible and secondary structural properties indicative of correctly folded flavoproteins. Anaerobic redox titrations on the individual domains assisted in assignment of the midpoint potentials for the high- and low-potential flavin. For the isolated FMN domain, the midpoint potentials for the oxidized/semiquinone (ox/sq) couple and semiquinone/hydroquinone (sq/hq) couple are -112 and -221 mV, respectively, at pH 7.0 and 25 degrees C. The corresponding couples in the isolated FAD domain are -222 mV (ox/sq) and -288 mV (sq/hq). Both flavins form blue neutral semiquinone species characterized by broad absorption peaks in the long-wavelength region during anaerobic titration with sodium dithionite. In full-length MSR, the values of the FMN couples are -109 mV (ox/sq) and -227 mV (sq/hq), and the corresponding couple values for FAD are -254 mV (ox/sq) and -291 mV (sq/hq). Separation of the MSR flavins does not perturb their thermodynamic properties, as midpoint potentials for all four couples are similar in isolated domains and in full-length MSR. The redox properties of MSR are discussed in relation to other members of the diflavin oxidoreductase family and the mechanism of electron transfer.

Binding Sites↗

PubMed2003

Extensive conformational sampling in a ternary electron transfer complex.

Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.

Amino Acid Sequence↗

PubMed2003

Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode. Part 1. Wild-type enzyme redox mediation.

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe-4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe-4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k(s) = 0.93 x 10(5) M(-1) s(-1)) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0-1.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA-N per lOOg wet fish muscle). The obtained results compared well with a reference method based on picric acid.

Animals↗

PubMed2003

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.

2,6-Dichloroindophenol↗

PubMed2002

Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry.

Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxidative half-reactions using dimethylglycine and O2 as substrates. The reductive half-reaction is triphasic. The rate of the fast phase is dependent on substrate concentration, involves flavin reduction, and has a limiting rate constant of 244 s(-1). This phase also displays a kinetic isotope effect of 2.9. Completion of the first kinetic phase generates an intermediate with broad spectral signature between 350 and 500 nm, which is attributed to a reduced enzyme-iminium charge-transfer species, similar to the purple intermediate that accumulates in reactions of D-amino acid oxidase (DAAO) with alanine. The second phase (16 s(-1)) is independent of substrate concentration and is attributed to iminium hydrolysis/deprotonation. The third phase (2 s(-1)) is attributed to product release, the rate of which is less than the steady-state turnover rate (10.6 s(-1)). Flavin oxidation of dithionite- and dimethylglycine-reduced enzyme by O2 occurs in a single phase, and the rate shows a linear dependence on oxygen concentration, giving bimolecular rate constants of 342 and 201 mM(-1) x s(-1), respectively. Enzyme-monitored turnover experiments indicate that decay of the reduced enzyme-iminium intermediate is rate-limiting, consistent with rate constants determined from single turnover studies. A minimal kinetic mechanism is presented, which establishes a close relationship to the mechanism of action of DAAO. The covalent flavin in dimethylglycine oxidase is identified as an alphaN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics.

Arthrobacter↗

PubMed2002

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.

Amino Acid Substitution↗