Biomedical subjects

Lee M Hoffart

Publications and source records attributed to Lee M Hoffart.

8 recordsLinked to original sources

PubMed2006

Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase.

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, alphaKG, to CO(2) and succinate. Our recent dissection of the mechanism of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, revealed that two transient complexes accumulate during catalysis in the presence of saturating substrates. The first complex contains the long-postulated C-H-cleaving Fe(IV)-oxo intermediate, J, and the second is an enzyme.product(s) complex. Here, we demonstrate the accumulation of two transient complexes in the reaction of a prolyl-4-hydroxylase (P4H), a functional homologue of human alphaKG-dependent dioxygenases with essential roles in collagen biosynthesis and oxygen sensing. The kinetic and spectroscopic properties of these two P4H complexes suggest that they are homologues of the TauD intermediates. Most notably, the first exhibits optical absorption and Mössbauer spectra similar to those of J and, like J, a large substrate deuterium kinetic isotope on its decay. The close correspondence of the accumulating states in the P4H and TauD reactions supports the hypothesis of a conserved mechanism for substrate hydroxylation by enzymes in this family.

Absorption↗

PubMed2006

Evidence for basic ferryls in cytochromes P450.

Using a combination of Mössbauer spectroscopy and density functional calculations, we have determined that the ferryl forms of P450(BM3) and P450cam are protonated at physiological pH. Density functional calculations were performed on large active-site models of these enzymes to determine the theoretical Mössbauer parameters for the ferryl and protonated ferryl (Fe(IV)OH) species. These calculations revealed a significant enlargement of the quadrupole splitting parameter upon protonation of the ferryl unit. The calculated quadrupole splittings for the protonated and unprotonated ferryl forms of P450(BM3) are DeltaE(Q) = 2.17 mm/s and DeltaE(Q) = 1.05 mm/s, respectively. For P450cam, they are DeltaE(Q) = 1.84 mm/s and DeltaE(Q) = 0.66 mm/s, respectively. The experimentally determined quadrupole splittings (P450(BM3), DeltaE(Q) = 2.16 mm/s; P450cam, DeltaE(Q) = 2.06 mm/s) are in good agreement with the values calculated for the protonated forms of the enzymes. Our results suggest that basic ferryls are a natural consequence of thiolate-ligated hemes.

Absorptiometry, Photon↗

PubMed2006

Evidence for two ferryl species in chloroperoxidase compound II.

Using a combination of density functional calculations and Mössbauer spectroscopy, we have examined chloroperoxidase compound II (CPO-II). The Mössbauer spectrum of CPO-II suggests the presence of two distinct ferryl species in an approximately 70:30 ratio. Density functional calculations and cryogenic reduction and annealing experiments allow us to assign the major species as an Fe(IV)OH intermediate. The Mössbauer parameters of the minor component are indicative of an authentic iron(IV)oxo species, but we have found the 70:30 ratio to be pH invariant. The unchanging ratio of component concentrations is in agreement with CPO-II's visible absorption spectrum, which shows no change over the enzyme's range of pH stability.

Chloride Peroxidase↗

PubMed2006

A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.

Animals↗

PubMed2006

Oxygen activation by a mixed-valent, diiron(II/III) cluster in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) catalyzes the ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate (myo-inositol, MI) to d-glucuronate (DG). The preceding paper [Xing, G., Hoffart, L. M., Diao, Y., Prabhu, K. S., Arner, R. J., Reddy, C. C., Krebs, C., and Bollinger, J. M., Jr. (2006) Biochemistry 45, 5393-5401] demonstrates by Mössbauer and electron paramagnetic resonance (EPR) spectroscopies that MIOX can contain a non-heme dinuclear iron cluster, which, in its mixed-valent (II/III) and fully oxidized (III/III) states, is perturbed by binding of MI in a manner consistent with direct coordination. In the study presented here, the redox form of the enzyme that activates O(2) has been identified. l-Cysteine, which was previously reported to accelerate turnover, reduces the fully oxidized enzyme to the mixed-valent form, and O(2), the cosubstrate, oxidizes the fully reduced form to the mixed-valent form with a stoichiometry of one per O(2). Both observations implicate the mixed-valent, diiron(II/III) form of the enzyme as the active state. Stopped-flow absorption and freeze-quench EPR data from the reaction of the substrate complex of mixed-valent MIOX [MIOX(II/III).MI] with limiting O(2) in the presence of excess, saturating MI reveal the following cycle: (1) MIOX(II/III).MI reacts rapidly with O(2) to generate an intermediate (H) with a rhombic, g < 2 EPR spectrum; (2) a form of the enzyme with the same absorption features as MIOX(II/III) develops as H decays, suggesting that turnover has occurred; and (3) the starting MIOX(II/III).MI complex is then quantitatively regenerated. This cycle is fast enough to account for the catalytic rate. The DG/O(2) stoichiometry in the reaction, 0.8 +/- 0.1, is similar to the theoretical value of 1, whereas significantly less product is formed in the corresponding reaction of the fully reduced enzyme with limiting O(2). The DG/O(2) yield in the latter reaction decreases as the enzyme concentration is increased, consistent with the hypothesis that initial conversion of the reduced enzyme to the MIOX(II/III).MI complex and subsequent turnover by the mixed-valent form is responsible for the product in this case. The use of the mixed-valent, diiron(II/III) cluster by MIOX represents a significant departure from the mechanisms of other known diiron oxygenases, which all involve activation of O(2) from the II/II manifold.

Animals↗

PubMed2006

Evidence for C-H cleavage by an iron-superoxide complex in the glycol cleavage reaction catalyzed by myo-inositol oxygenase.

myo-Inositol oxygenase (MIOX) activates O2 at a mixed-valent nonheme diiron(II/III) cluster to effect oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate [myo-inositol (MI)] by four electrons to d-glucuronate. Abstraction of hydrogen from C1 by a formally (superoxo)diiron(III/III) intermediate was previously proposed. Use of deuterium-labeled substrate, 1,2,3,4,5,6-[2H]6-MI (D6-MI), has now permitted initial characterization of the C-H-cleaving intermediate. The MIOX.1,2,3,4,5,6-[2H]6-MI complex reacts rapidly and reversibly with O2 to form an intermediate, G, with a g = (2.05, 1.98, 1.90) EPR signal. The rhombic g-tensor and observed hyperfine coupling to 57Fe are rationalized in terms of a (superoxo)diiron(III/III) structure with coordination of the superoxide to a single iron. G decays to H, the intermediate previously detected in the reaction with unlabeled substrate. This step is associated with a kinetic isotope effect of > or =5, showing that the superoxide-level complex does indeed cleave a C-H(D) bond of MI.

Animals↗

PubMed2005

Kinetic dissection of the catalytic mechanism of taurine:alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli.

Recent studies on taurine:alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli have provided evidence for a three-step, minimal kinetic mechanism involving the quaternary TauD.Fe(II).alpha-ketoglutarate.taurine complex, the taurine-hydroxylating Fe(IV)-oxo intermediate (J) that forms upon reaction of the quaternary complex with O(2), and a poorly defined, Fe(II)-containing intermediate state that converts in the rate-limiting step back to the quaternary complex [Price, J. C., Barr, E. W., Tirupati, B., Bollinger, J. M., Jr., and Krebs, C. (2003) Biochemistry 42, 7497-7508]. The mapping of this kinetic mechanism onto the consensus chemical mechanism for the Fe(II)- and alpha-ketoglutarate-dependent engendered several predictions and additional questions that have been experimentally addressed in the present study. The results demonstrate (1) that postulated intermediates between the quaternary complex and J accumulate very little or not at all; (2) that decarboxylation of alpha-ketoglutarate occurs prior to or concomitantly with formation of J; (3) that the second intermediate state comprises one or more product complex with Mossbauer features that are partially resolved from those of the binary TauD.Fe(II), ternary TauD.Fe(II).alpha-ketoglutarate, and quaternary TauD.Fe(II).alpha-ketoglutarate.taurine complexes; and (4) that the rate-determining step in the catalytic cycle is release of product(s) prior to the rapid, ordered binding of alpha-ketoglutarate and then taurine to regenerate the O(2)-reactive quaternary complex. The results thus integrate the previously proposed kinetic and chemical mechanisms and indicate which of the postulated intermediates in the latter will be detectable only upon perturbation of the kinetics by changes in reaction conditions (e.g., temperature), protein mutagenesis, the use of substrate analogues, or some combination of these.

Binding Sites↗

PubMed2005

Escherichia coli quinolinate synthetase does indeed harbor a [4Fe-4S] cluster.

Quinolinic acid is an intermediate in the biosynthesis of nicotinamide-containing redox cofactors. The ultimate step in the formation of quinolinic acid in prokaryotes is the condensation of iminosuccinate and dihydroxyacetone phosphate, which is catalyzed by the product of the nadA gene in Escherichia coli. A combination of UV-vis, Mössbauer, and EPR spectroscopies, along with analytical methods for the determination of iron and sulfide, demonstrates for the first time that anaerobically purified quinolinate synthetase (NadA) from E. coli contains one [4Fe-4S] cluster per polypeptide. The protein is active, catalyzing the formation of quinolinic acid with a Vmax [ET]-1 of 0.01 s-1.

Azotobacter vinelandii↗