Biomedical subjects

Steven O Mansoorabadi

Publications and source records attributed to Steven O Mansoorabadi.

7 recordsLinked to original sources

PubMed2006

Analysis of the Cob(II)alamin-5'-deoxy-3',4'-anhydroadenosyl radical triplet spin system in the active site of diol dehydrase.

A triplet spin system (S=1) is detected by low-temperature electron paramagnetic resonance (EPR) spectroscopy in samples of diol dehydrase and the functional adenosylcobalamin (AdoCbl) analogue 5'-deoxy-3',4'-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence and absence of the substrate (R,S)-1,2-propanediol. In both cases, the spectra include a prominent half-field transition (DeltaM(S) = 2) that is a hallmark of strongly coupled triplet spin systems. The appearance of 59Co hyperfine splitting in the EPR signals and the positions (g values) of the signals in the spectra show that half of the triplet spin is contributed by the low-spin Co2+ of cob(II)alamin. Line width effects from isotopic labeling (13C and 2H) in the 5'-deoxy-3',4'-anhydroribosyl ring demonstrate that the other half of the spin triplet is from an allylic 5'-deoxy-3',4'-anhydroadenosyl (anhydroadenosyl) radical. The zero-field splitting (ZFS) tensors describing the magnetic dipole-dipole interactions of the component spins of the triplets have rhombic symmetry because of electron spin delocalization within the organic radical component and the proximity of the radical to the low-spin Co2+. The dipole-dipole interaction was modeled as a summation of point-dipole interactions involving the spin-bearing orbitals of the anhydroadenosyl radical and cob(II)alamin. Geometries which are consistent with the ZFS tensors in the presence and absence of the substrate position the 5'-carbon of the anhydroadenosyl radical 3.5 and 4.1 A from Co2+, respectively. Homolytic cleavage of the cobalt-carbon bond of the analogue in the absence of the substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens the bond prior to binding of the substrate.

Binding Sites↗

PubMed2006

Reaction of adenosylcobalamin-dependent glutamate mutase with 2-thiolglutarate.

We have investigated the reaction of glutamate mutase with the glutamate analogue, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with a Ki of 0.05 mM. However, rather than simply binding inertly at the active site, 2-thiolglutarate elicits cobalt-carbon bond homolysis and the formation of 5'-deoxyadenosine. The enzyme exhibits a complicated EPR spectrum in the presence of 2-thiolglutarate that is markedly different from any previously observed with the enzyme. The spectrum was simulated well by assuming that it arises from electron-electron spin coupling between a thioglycolyl radical and low-spin Co2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical approximately 10 A from the cobalt and at a tilt angle of approximately 70 degrees to the normal of the corrin ring. This orientation is in good agreement with that expected from the crystal structure of glutamate mutase complexed with the substrate. 2-Thiolglutarate appears to react in a manner analogous to that of glutamate by first forming a thiolglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfur-stabilized thioglycolyl radical. The thioglycolyl radical accumulates on the enzyme, suggesting it is too stable to undergo further steps in the mechanism at a detectable rate.

Cobamides↗

PubMed2006

EPR spectroscopic and computational characterization of the hydroxyethylidene-thiamine pyrophosphate radical intermediate of pyruvate:ferredoxin oxidoreductase.

The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at X-band and D-band microwave frequencies. EPR spectra, obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14N/15N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) pi-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1H-hyperfine splittings from the methyl group of pyruvate and the 13C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, sigma/n-type cation radical.

Electron Spin Resonance Spectroscopy↗

PubMed2006

Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle.

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.

Catalysis↗

PubMed2005

Structure and catalytic properties of an engineered heterodimer of enolase composed of one active and one inactive subunit.

Enolase is a dimeric enzyme that catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. This reversible dehydration is effected by general acid-base catalysis that involves, principally, Lys345 and Glu211 (numbering system of enolase 1 from yeast). The crystal structure of the inactive E211Q enolase shows that the protein is properly folded. However, K345 variants have, thus far, failed to crystallize. This problem was solved by crystallization of an engineered heterodimer of enolase. The heterodimer was composed of an inactive subunit that has a K345A mutation and an active subunit that has N80D and N126D surface mutations to facilitate ion-exchange chromatographic separation of the three dimeric species. The structure of this heterodimeric variant, in complex with substrate/product, was obtained at 1.85 A resolution. The structure was compared to a new structure of wild-type enolase obtained from crystals belonging to the same space group. Asymmetric dimers having one subunit exhibiting two of the three active site loops in an open conformation and the other in a conformation having all three loops closed appear in both structures. The K345A subunit of the heterodimer is in the loop-closed conformation; its Calpha carbon atoms closely match those of the corresponding subunit of wild-type enolase (root-mean-squared deviation of 0.23 A). The kcat and kcat/Km values of the heterodimer are approximately half those of the N80D/N126D homodimer, which suggests that the subunits in solution are kinetically independent. A comparison of enolase structures obtained from crystals belonging to different space groups suggests that asymmetric dimers can be a consequence of the asymmetric positioning of the subunits within the crystal lattice.

Catalysis↗

PubMed2005

Characterization of a succinyl-CoA radical-cob(II)alamin spin triplet intermediate in the reaction catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase.

The electron paramagnetic resonance (EPR) spectrum of an intermediate freeze trapped during the steady state of the reaction catalyzed by the adenosylcobalamin (AdoCbl)-dependent enzyme, methylmalonyl-CoA mutase, has been studied. The EPR spectrum is that of a hybrid triplet spin system created as a result of strong electron-electron spin coupling between an organic radical and the low-spin Co(2+) in cob(II)alamin. The spectrum was analyzed by simulation to obtain the zero-field splitting (ZFS) parameters and Euler angles relating the radical-to-cobalt interspin vector to the g axis system of the low-spin Co(2+). Labeling of the substrate with (13)C and (2)H was used to probe the identity of the organic radical partner in the triplet spin system. The patterns of inhomogeneous broadening in the EPR signals produced by [2'-(13)C]methylmalonyl-CoA and [2-(13)C]methylmalonyl-CoA as well as line narrowing resulting from deuterium substitution in the substrate were consistent with those expected for a succinyl-CoA radical wherein the unpaired electron was centered on the carbon alpha to the free carboxyate group of the rearranged radical. The interspin distance and the Euler angles were used to position this product radical into the active site of the enzyme.

Acyl Coenzyme A↗

PubMed2003

The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes.

The radical intermediates generated during the catalytic cycles of adenosylcobalamin-dependent enzymes occur in pairs. The positions of radicals residing on the cofactor, substrate or protein, relative to the position of the low-spin Co(2+) from the cob(II)alamin intermediate, can be extracted from electron paramagnetic resonance (EPR) spectra of the spin-coupled pairs. Examples of radical-Co(2+) pairs that span a range of interspin distances from 3 to 13A have been presented. Interspin distances greater than 5A require motion of one or more of the participating species. EPR spectroscopy provides a convenient means to determine the structures of these transient intermediates.

Binding Sites↗